Purified protein

ABSTRACT

Described herein are methods for purifying protein, and more particularly to methods for purifying protein that minimize the development of undesirable odors and flavors in the purified protein and increase protein yield.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.16/138,876, filed on Sep. 21, 2018, which claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62/562,298, filed onSep. 22, 2017, now U.S. Pat. No. 11,051,532, and U.S. Provisional PatentApplication No. 62/670,478, filed on May 11, 2018, the entire contentsof which are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to methods for purifying protein, and moreparticularly to methods for purifying protein that minimize thedevelopment of undesirable odors and flavors in the purified protein,enhance functionality and increase protein yield. This invention alsorelates to food products including purified protein.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an ASCII text file named “38767-0119002_SL”. The ASCIItext file, created on Jun. 3, 2021, is 46.3 kilobytes in size. Thematerial in the ASCII text file is hereby incorporated by reference inits entirety.

BACKGROUND

The success of food products that mimic animal derived food products(e.g., cheese or meat) is largely dependent on generating functionalprotein that can be manipulated and has low-flavor so the source of theprotein is not readily identifiable and does not provide any off-flavorsto the food product. Common protein purification methods typicallyinclude steps with chemicals that are not food-safe and/or that resultin denatured protein. It would be useful to have a method of proteinpurification that is food-safe and results in minimal undesirable odorsand flavors in the purified protein.

SUMMARY

This document provides protein compositions, and it also providesmethods for purifying protein from microbial cells including eukaryotes,fungi, prokaryotes, and Archaea cells, using at least a pH of about 8.5throughout the process, which results in a protein composition. Thisdocument also provides food products that include these proteincompositions. In some embodiments, the methods described herein arefood-safe, inexpensive, and scalable, while minimizing the developmentof undesirable odors and flavors in the purified protein and increasingprotein yield. In some embodiments of methods of purifying proteinprovided herein in which the pH is less than 8.5 (e.g., 8.0 or less)during the purification process, increased off-flavors and/or off-odorscan be present in the resulting protein composition and process yieldscan be reduced compared otherwise corresponding methods of purifyingprotein in which the pH is greater than 8.5 (e.g., 9.0 or greater)during some or all of the purification process. In some embodiments,compositions described herein can be food-safe and inexpensive, withminimal undesirable odors and flavors. Total cellular protein, e.g.,proteins that are isolated from throughout a cell or produced by it,including proteins from the cytoplasm, and nucleus and subcellularcompartments (e.g., lysosomes, peroxisomes, mitochondria, endoplasmicreticulum, Golgi apparatus, periplasm, secretory vesicles, extracellularmatrix, biofilm, chloroplast and nucleus) as applicable, can be purifiedusing the methods described herein. In some embodiments, a proteincomposition as described herein can comprise total cellular protein, butthe term “total” does not indicate that every cellular protein ispresent in the protein composition. In some embodiments, a proteincomposition as described herein can consist essentially of totalcellular protein.

Furthermore, the protein in a protein composition can be functional. Asdescribed herein, functional proteins can have one or more of thefollowing properties: non-denatured; capable of forming a gel uponheating (e.g., a suspension of about 25 to about 250 mg/mL (e.g., about25 to about 50 mg/mL, about 25 to about 100 mg/mL, about 25 to about 150mg/mL, about 25 to about 200 mg/mL, about 50 to about 250 mg/mL, about100 to about 250 mg/mL, about 150 to about 250 mg/mL, or about 200 toabout 250 mg/mL) at a pH of about 7.0) thermally transitions to a gelupon heating to about 65° C.); thermally denatures during incubationbetween about 50° C. and about 85° C., with greater than about 80% ofthe protein denaturing after about 20 minutes at about 85° C., asmeasured either by differential scanning calorimetry (DSC) ordifferential scanning fluorimetry (DSF); in a solution or suspension ofpurified protein at or above about 50 mg/mL (5% w/v), protein forms afreestanding gel (with, e.g., a 100 Pa storage modulus) when heated ator above about 85° C. for about 20 minutes; can denature and gel betweenabout pH 5.5 and about pH 10.0; can denature and gel in solutions withionic strength (I) below about 0.5M, when I is calculated based on theconcentration of non-protein solutes; at a protein concentration ofabout 10 mg/mL, particle size distribution D10, D50 and D90 are lessthan about 0.1 μm, 1.0 μm and 5 μm, respectively; has enzymaticactivity; has an emulsion activity index (EAI) of greater than or equalto about 50 m²/g protein across a pH range of about 4.0 to about 8.0.

In some embodiments, a (w/v) suspension can refer to the amount of drysolids (in grams) per 100 mL of solution.

Non-limiting examples of functional proteins that can be present in aprotein composition include proteins that have enzymatic activity suchas, without limitation, cysteine synthase (Met17p, ED 2.5.1.47),cystathionine beta-synthase (Cys4p, EC 4.2.1.22), hexokinase, glucoseoxidase, glutathione reductase, catalase, and lipase.

Enzymatic activity also can be described more generically and examplescan include, for example amino acid catabolism (e.g., hydrogen sulfide(H₂S) present at less than about 0.1 ppm in the headspace when noL-cysteine (e.g., L-cysteine by itself, or provided in the form of amixture of isomers) is added (e.g., to 5 mL of a 2% (w/v) suspension atpH 7.0), and H₂S present at greater than or equal to about 0.2 ppm(e.g., greater than or equal to about 0.3 ppm) after (e.g., after about24 hours at 25° C.) L-cysteine is added to 25 mM final concentration(e.g., to 5 mL of a 2% (w/v) suspension)), glucose catabolism (e.g.,generation of pyruvate from glucose, generation of glucose-6-phosphate,generation of lactate, production of D-glucono-δ-lactone), lipidcatabolism (e.g., lipid hydrolysis), reduction of glutathione disulfide,and decomposition of hydrogen peroxide For example, enzymatic activitycan be illustrated using a single-enzyme reaction (e.g., generation ofglucose-6-phosphate from glucose by hexokinase) or a multi-enzymereaction, e.g., transformation of a starting material to a final productby more than one enzyme (e.g., generation of pyruvate from glucose bythe enzymes of glycolysis or generation of glutathione from glutamate,cysteine and glycine by the cellular glutathione biosynthesis pathway).

The protein composition can have food activity. As described herein,proteins with food activity can have one or more of the followingproperties (defined on a per-gram basis): capable of forming a gel;capable of emulsifying oil and water (oil-in water, water-in-oil);capable of emulsifying air and water (air-in-water).

In the description and Figures, the following abbreviations are used: CS(cell suspension); RN (cell wash); LY (lysate, e.g., obtained by beadmilling, homogenizer, high shear mixer or microfluidizer); CN (centrate,e.g., supernatant of centrifugal spin to remove solids); MF(microfiltration, e.g., using a pore size of 0.2. 0.3, or 0.45 μm indiameter); DF (diafiltration, e.g., using pH 9.3+1-0.3 water); UF(ultrafiltration, e.g., using a molecular weight cutoff of 5, 10, 30, or50 kDa); PZ (pasteurization, e.g., at 65° C. for 60 seconds); SD (spraydrying, e.g., with an inlet temperature of 180° C., an outlettemperature of 80° C. and a feed of 0.27 LPM).

The term “about” is used with respect to a particular value to accountfor experimental variation when measuring the value.

In one aspect, this document includes a method for purifying proteinfrom a cell (e.g., a plurality of cells), the method including lysing anaqueous suspension of the plurality of cells to obtain a cell lysate;clarifying the cell lysate, optionally in the presence of one or moreflocculants, to obtain a clarified lysate; filtering the clarifiedlysate to obtain a filtered lysate; concentrating the filtered lysate toobtain a protein composition; and optionally pasteurizing the proteincomposition of protein to obtain a pasteurized protein composition,wherein the lysing, clarifying, and filtering steps, independently, areperformed at a pH between about 8.5 and about 12.0.

In some embodiments of a protein composition or a food product describedherein, the protein composition or food product contains less than 10%by weight animal products. In some embodiments of a protein compositionor a food product described herein, the protein composition or foodproduct contains less than 5% by weight animal products. In someembodiments, a protein composition or food product described hereincontains less than 1% by weight animal products. In some embodiments, aprotein composition or food product described herein contains no animalproducts.

In some embodiments of a protein composition or a food product describedherein, the protein composition or food product contains less than 10%by weight animal-derived products. In some embodiments of a proteincomposition or a food product described herein, the protein compositionor food product contains less than 5% by weight animal-derived products.In some embodiments, a protein composition or a food product describedherein contains less than 1% by weight animal-derived products. In someembodiments, a protein composition or food product described hereincontains no animal-derived products.

In some embodiments of a protein composition or a food product describedherein, the protein composition or food product contains less than 10%by weight animal meat. In some embodiments of a protein composition or afood product described herein, the protein composition or food productcontains less than 5% by weight animal meat. In some embodiments, aprotein composition or food product described herein contains less than1% by weight animal meat. In some embodiments, a protein composition orfood product described herein contains no animal meat.

In some embodiments of a protein composition or a food product describedherein, the protein composition or food product is free of or issubstantially free of lactose, E. coli, whey, casein, animal fat, soyproteins, nut proteins, ovalbumins, gelatin, dairy products, animalproducts, animal-derived products, agar, carrageenan, tofu, cholesterol,or two or more thereof.

As used herein, the term “animal products” refers to a material obtainedfrom or produced by the body of an animal (e.g., a mammal, a bird, afish, an amphibian, a reptile, an insect, a mollusk, a crustacean, acoral, an arachnid, a velvet worm, or a horseshoe crab). Examplesinclude, without limitation, meat, fat, flesh, blood, milk, eggs,isinglass, rennet, fur, skin, hair, bone, fibers, cartilage, casein,gelatin, and honey. The term “no animal products” means that thecomposition does not contain any animal products.

As used herein, the term “animal-derived products” refers to a materialor compound derived from the body of an animal (e.g., a mammal, a bird,a fish, an amphibian, a reptile, an insect, a mollusk, a crustacean, acoral, an arachnid, a velvet worm, or a horseshoe crab). Examplesinclude, without limitation, a material or compound derived from animalmeat, fat, flesh, blood, milk, eggs, isinglass, rennet, fur, skin, hair,bone, fibers, cartilage, casein, gelatin, and honey. Further examples ofanimal-derived products include materials isolated from the body of ananimal, including without limitation, hormones, amino acids, vitamins,organic acids, proteins, collagen, dyes, fatty acids, oils, glycerol,sugars, keratin, and nucleic acids isolated from the body of an animal.The term “no animal-derived products” means that the composition doesnot contain any animal products.

As used herein, the term “animal meat” refers to a flesh of an animal(e.g., a mammal, a bird, a fish, an amphibian, a reptile, an insect, amollusk, a crustacean, a coral, an arachnid, a velvet worm, or ahorseshoe crab). Examples include without limitation, muscle and organs.The term “no animal meat” means that the composition does not containany animal meat.

As used herein, the term “substantially free of” means less than 5.0% byweight, (e.g., less than 5.0% by weight, less than 4.0% by weight, lessthan 3.0% by weight, less than 2.5% by weight, less than 2.0% by weight,less than 1.5% by weight, less than 1.0% by weight, less than 0.5% byweight, less than 0.1% by weight or less than 0.01% by weight) of thereferenced ingredient is present in a composition. For example, a dairyreplica as disclosed herein in is substantially free of animal productswhen it contains less than 5.0% by weight (e.g., less than 4.0% byweight, less than 3.0% by weight, less than 2.5% by weight, less than2.0% by weight, less than 1.5% by weight, less than 1.0% by weight, lessthan 0.5% by weight, less than 0.1% by weight, or less than 0.01% byweight) of animal products.

As used herein, the term “free of” means that none of the referencedingredient is detectable in a composition. For example, a proteincomposition or food product disclosed herein in is free of animalproducts when it contains no detectable animal products.

As used herein a “bacteria-derived protein”, “yeast-derived protein”,“algae-derived protein”, “fungus-derived protein”, or “plant-derivedprotein” refers to the immediate production organism of the protein, andcan mean any protein that is produced in a bacterium, a yeast, an algae,a fungus, or a plant, independently of whether the protein is nativelyexpressed in the bacterium, yeast, algae, fungus, or plant,respectively.

The term “not natively expressed” can refer to a protein that isproduced in an organism that does not produce said protein in nature.Non-limiting examples of a protein that is not natively expressedinclude an animal protein expressed in bacteria, a plant proteinexpressed in yeast, and an animal protein expressed in algae.

The term “pasteurized” can mean any process, treatment, or combinationthereof, that is applied to food to reduce the most resistantmicroorganism(s) of public health significance to a level that is notlikely to present a public health risk under normal conditions ofdistribution and storage.

The term “aromaome” can mean the totality of aromas associated by theordinary human observer with a particular food, ingredient, or cookingprocess. Non-limiting examples of aromaomes are a poultry aromaome, achicken aromaome, a beef aromaome, a pork aromaome, a seafood aromaome,a game meat aromaome, a cinnamon aromaome, a chocolate aromaome, a deepfrying aromaome, and a grilling aromaome.

In one aspect, this disclosure includes a method for purifying proteinfrom a plurality of cells, the method including lysing an aqueoussuspension of the plurality of cells to obtain a cell lysate; clarifyingthe cell lysate, optionally in the presence of one or more flocculants,to obtain a clarified lysate; filtering the clarified lysate to obtain aprotein composition; and optionally pasteurizing the proteincomposition, to obtain a pasteurized protein composition, wherein stepsa), b), c), and d) independently, are performed at a pH between about8.5 and about 12.0.

In another aspect, this disclosure includes a method for purifyingprotein from a plurality of cells, the method including lysing anaqueous suspension of the plurality of cells to obtain a cell lysate;filtering the cell lysate to obtain a protein composition; andoptionally pasteurizing the protein composition to obtain a pasteurizedprotein composition, wherein steps a), b), and c) independently, areperformed at a pH between about 8.5 and about 12.0.

These and other embodiments can optionally include any of the following.Filtering can include microfiltration, ultrafiltration, diafiltration,or a combination thereof. A clarifying step can be performed bycentrifugation to less than about 20% dry solids. A plurality of cellscan include microbial cells. A method can further include washing anaqueous suspension of a plurality of cells at a pH between about 8.5 andabout 12.0 before step a). A protein composition can include at leastabout 35%, on a dry weight basis, of compounds larger than 5 kDa. Atleast about 50% of the protein in a protein composition can fall betweenabout 10 kDa and about 200 kDa.

In an another aspect, this disclosure includes protein compositionproduced by a method comprising: lysing an aqueous suspension of aplurality of cells to obtain a cell lysate; filtering the cell lysate toobtain a protein composition; and optionally pasteurizing the proteincomposition to obtain a pasteurized protein composition, wherein stepsa), b), and c) independently, are performed at a pH between about 8.5and about 12.0.

This and other embodiments can optionally include any of the following.Filtering can include microfiltration, ultrafiltration, diafiltration,or a combination thereof. A process can further include washing anaqueous suspension of a plurality of cells at a pH between about 8.5 andabout 12.0 before step a). At least about 50% of the protein in aprotein composition can fall between about 10 kDa and about 200 kDa. Aprotein composition can have a buffering capacity of less than about 2.5mmol NaOH per gram dry solids. Hydrogen sulfide (H₂S) can be detectablein an amount of less than about 0.1 ppm after about 24 hours at 25° C.when L-cysteine is not added to 5 mL of a 2% (w/v) suspension of aprotein composition at pH 7.0. Hydrogen sulfide can be detectable anamount of at least about 0.2 ppm about 24 hours at 25° C. after 5 mL ofa 2% (w/v) suspension of a protein composition is brought to about 25 mMfinal concentration of L-cysteine.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and drawings, and fromthe claims. The word “comprising” in the claims may be replaced by“consisting essentially of” or with “consisting of,” according tostandard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of process variants for isolation andconcentration of protein.

FIG. 2 is a schematic of process variants for isolation andconcentration of protein. All steps may be followed by pasteurization(PZ) and/or spray drying (SD). Process conditions in all: pH 9.3+/−0.3;temperature <10° C.

FIG. 3 is a plot showing that the product of high pH process createsfirmer gels when heated. Process Variant A (see FIG. 2 ) was conductedat pH 6.5 or at pH 9.5. Rheology of the resulting material was measuredusing a hybrid rheometer. Vertical axis shows storage modulus (Pa) inlog scale. Horizontal axis shows incubation temperature.

FIG. 4 is a plot showing that removing small molecules from microbialprotein isolates and concentrates increased gel firmness by >5×; theeffect was independent of solids removal. Process Variants B and C (seeFIG. 2 ) were conducted at pH 9.3. In-process samples were taken ofcentrate (“CN”) or lysate (“LY”) and the final product of respectiveprocess B (“CN/UFO”) or process C (“LY/UFO”). Each sample was freezedried, then suspended to 10% (w/v) in MILLI-Q® water. Suspensions wereassayed at pH 7.5. Rheology of the resulting material was measured usinga hybrid rheometer. Vertical axis shows storage modulus (Pa) in logscale. Horizontal axis shows incubation temperature.

FIG. 5 is a plot showing that reconstituting protein isolates andconcentrates with native small molecules reduces gel firmness by >2×.Process Variant C (see FIG. 2 ) was conducted at pH 9.3. The finalmaterial was freeze dried, then resuspended to 10% (w/v) in eitherMILLI-Q® water or in the initial UF permeate taken as an in-processsample. Suspensions were assayed at pH 7.5. Rheology of the resultingmaterial was measured using a hybrid rheometer. Vertical axis showsstorage modulus (Pa) in log scale. Horizontal axis shows incubationtemperature.

FIG. 6 is a plot showing the absence of detectable hydrogen sulfide(H₂S) in the headspace and the appearance of >0.1 ppm hydrogen sulfide(I12S) after addition of 25 mM L-cysteine to microbial proteincompositions prepared using Process Variants C or Process Variant D.Microbes used to prepare the protein compositions are representatives ofeither Bacteria (Escherichia coli) or Eukarya (Pichia pastoris orSaccharomyces cerevisiae).

FIG. 7 is a photograph of the results of assaying for H₂S in theheadspace of lysates from Saccharomyces cells were prepared at about 2%(w/v) final by taking an in-process sample after homogenizer lysisduring Process Variant C. The upper half shows standards, and the lowerhalf shows the results of the lysate with no added cysteine (left),Process Variant C with added pyridoxal hydrochloride (center), andProcess Variant C with added L-cysteine (right).

FIG. 8 is a structure of Heme B.

DETAILED DESCRIPTION

Proteins in their undenatured state can contribute to the success offood replica products, such as meat and dairy replica products. Existingcommercial protein extraction processes however, can result indenaturation of such proteins. Further, many proteins that might havefunctionality in food replica products have associated colors or aromas,which can detract from or inhibit their application.

In general, this document provides protein compositions as well asmethods and materials for purifying total cellular protein from cellsresulting in protein compositions that can be used, for example, in foodreplicas, e.g., meat and dairy replica products or substitutes.

Methods for Producing Protein Compositions

In some embodiments, a protein composition can be purified using any ofthe methods described herein). Suitable cells from which proteins can beextracted include, without limitation, cells from fungi, algae,prokaryotes, and Archea. In some embodiments, suitable cells may benaturally found in single-celled organisms (including yeasts) or inmulticellular organisms (including Ascomycota and Basidiomycota). Insome embodiments, a protein composition can be purified from one or morefungal species from, for example, the genera Saccharomyces, Pichia,Candida, Hansenula, Torulopsis, Kluyveromyces, Yarrowia, Aspergillus,Trichoderma, or Fusarium. For example, a protein composition can bepurified from Saccharomyces cerevisiae, Pichia pastoris, Candidaboidinii, Hansenula polymorpha, Kluyveromyces lactis, Yarrowialipolytica, or Fusarium venenatum cells. In some embodiments, a proteincomposition can be purified from one or more archaeal or bacterialspecies from, for example, the genera Bacillus, Escherichia,Lactobacillus, Corynebacterium, Pseudomonas, or Methanococcus. Forexample, a protein composition can be purified from E. coli, Bacillussubtilis, Lactobacillus lactis, Corynebacterium glutamicum, Pseudomonasfluorescens, or Methanococcus maripaludis. In some embodiments, aprotein composition can be purified from one or more algal species from,for example, the genera Chlorella, Cyanobacteria, Euglenid, orSpirulina. For example, a protein composition can be purified fromChlorella protothecoides, Arthrospira platensis, Euglena gracilis, orNostoc flagelliforme.

In some embodiments, one or more proteins in a protein compositiondescribed herein can have functional activity as a biocatalyst, as afood processing aid, an enzyme, as a flavor enhancer, a therapeutic, ora nutraceutical.

In some embodiments, a protein composition or a protein purified from amicrobe can include one or more heterologous proteins (e.g., from aspecies different from the organism used to purify a protein or proteincomposition such as, for example, a protein from a eukaryote, an animal,a plant, an algae, a thermophile, a yeast, a bacteria, a protist or anarchea). In some embodiments, the heterologous protein has functionalactivity as a biocatalyst, as a food processing aid, an enzyme, as aflavor enhancer, a therapeutic, a sweetener, a pharmaceutical, or anutraceutical.

In some embodiments, an aqueous solution can include a buffer. Thebuffer can be any food-grade buffer (e.g., a buffer that includes sodiumphosphate, potassium phosphate, calcium phosphate, sodium acetate,potassium acetate, sodium citrate, calcium citrate, sodium bicarbonate,sodium lactate, potassium lactate, sodium malate, potassium malate,sodium gluconate, and/or potassium gluconate) at a concentration ofabout 2 mM to about 200 mM (e.g., about 2 mM to about 10 mM, about 10 mMto about 20 mM, about 10 mM to about 30 mM, about 20 mM to about 30 mM,about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 50 mM toabout 100 mM, or about 100 mM to about 200 mM), and a pH of about 8.5 toabout 12.0 (e.g., about 8.5 to about 9.0, about 9.0 to about 10.0, about9.0 to about 11.0, about 10.0 to about 11.0, about 11.0 to about 12.0,about 9.5 to about 10.5, about 9.5 to about 11.5, about 10.5 to about11.5, at 9.0, at 9.5, at 10.0, at 10.5, at 11.0, at 11.5, or at 12.0).

In some embodiments of the methods described herein, a plurality ofcells (e.g., microbial cells) can be suspended in an aqueous solution.In some embodiments, the plurality of cells can be washed. The pluralityof cells can be lysed at a pH between about 8.5 to about 12.0 (e.g.,about 8.5 to about 9.0, about 9.0 to about 10.0, about 9.0 to about11.0, about 10.0 to about 11.0, about 11.0 to about 12.0, about 9.5 toabout 10.5, about 9.5 to about 11.5, about 10.5 to about 11.5, at 9.0,at 9.5, at 10.0, at 10.5, at 11.0, at 11.5, or at 12.0) to obtain a celllysate. As described herein, maintaining a high pH during lysis can helpto improve lysis (e.g., protein yield) and/or clarification. Withoutlimitation, an aqueous suspension or a cell lysate can have from about2% to about 25% dry solids (i.e., the mass remaining after removing allwater). For example, an aqueous suspension or a cell lysate can havefrom about 2% to about 5%, about 5% to about 10%, about 10% to about15%, about 15% to about 20%, about 20% to about 25%, about 5% to about25%, about 10% to about 25%, about 15% to about 20%, about 2%, 5%, 7.5%,10%, 12.5%, 15%, 17.5%, 20%, 22.5%, or 25% dry solids. In someembodiments, lysis can be biochemical such as enzymatic cell walldegradation or the lysis can be chemical, e.g., surfactant-based lysis,chaotropic-based lysis, or organic solvent-based lysis. Additionally oralternatively, lysis also can be mechanical using, for example,sonication, bead milling, osmotic lysis, homogenization, manualgrinding, or by subjecting the cells to freeze-thaw cycles. Lysis can beperformed at a temperature between about 4° C. and about 15° C. (e.g.,about 4° C. to about 12° C., about 5° C. to about 10° C., about 4° C.,5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14°C. or 15° C.).

Purification of a cell lysate can include one or more steps of, forexample, centrifugation, clarification, precipitation, microfiltration,ultrafiltration, diafiltration (e.g., using a microfiltration orultrafiltration membrane), pasteurization, and/or spray drying. FIG. 1illustrates some exemplary schematics of different purification schemesthat may be employed. FIG. 2 illustrates four particular purificationschemes. In some embodiments, a protein composition can be a clarifiedlysate. In some embodiments, a protein composition can be a filtered(e.g., using one or more filtration steps) lysate, whether or not thelysate has been clarified. A protein composition can be used, e.g., infoods and food replica products.

A cell lysate can be optionally clarified by removing bulk solids,forming a clarified lysate. A variety of techniques can be used toclarify a cell lysate. For example, the cell lysate can be clarified bycentrifugation, gravity settling, or by adding diatomaceous earth.During clarification, in some embodiments, the pH is maintained at a pHbetween about 8.5 to about 12.0 (e.g., about 8.5 to about 9.0, about 9.0to about 10.0, about 9.0 to about 11.0, about 10.0 to about 11.0, about11.0 to about 12.0, about 9.5 to about 10.5, about 9.5 to about 11.5,about 10.5 to about 11.5, at 9.0, at 9.5, at 10.0, at 10.5, at 11.0, at11.5, or at 12.0). The cell lysate can be clarified to a dry solidscontent of less than 20%, e.g., less than 17%, 15%, 12%, 10%, 9%, 8%,7%, 6%, or 5% dry solids.

One or more flocculants can optionally be added to a final concentrationof about 0.1 to about 10 g/L to help improve the solids removal during aclarification step. Non-limiting examples of flocculants includealkylamine-epichlorohydrin, polydimethyldiallylammonium chloride, apolyamine (e.g., MAGNAFLOC®, SUPERFLOC®, or TRAMFLOC®, from BASF,Florham Park, N.J.), poly-ε-lysine, lime, hydrated lime, ferricchloride, ferric sulfate, ferrous sulfate, aluminum sulfate, sodiumaluminate, aluminium chloride, magnesium carbonate hydroxide, calciumcarbonate, calcium hydroxide, an activated silicate, a guar gum, astarch, a tannin, sodium alginate, polyaluminum sulfate, polyaluminumhydroxy chloride, BIO-FLOCK®, and a synthetic polyelectrolyte (e.g.,ZETAG®). In some embodiments, one or more flocculants are added. In someembodiments, a clarification step is performed without adding one ormore flocculants.

In some embodiments, a cell lysate can be optionally diluted using, forexample, water or an aqueous solution of salts or buffers, prior tosolids removal, while maintaining the pH between about pH 8.5 and 12.0.For example, a cell lysate can be diluted 1:1 with water. In someembodiments, one or more flocculants are added to a cell lysate and thecell lysate is diluted before clarification. In some embodiments, a celllysate is diluted before clarification, and the clarification stepproceeds without adding one or more flocculants. In some embodiments,one or more flocculants are added to a cell lysate and the cell lysateis not diluted before clarification. In some embodiments, aclarification step is performed without adding one or more flocculantsto a cell lysate and without diluting the cell lysate.

In some embodiments, a cell lysate (e.g., a cell lysate that has notundergone a clarification step, such as a clarification step asdescribed herein) can be filtered to obtain a filtered lysate. Afiltration step can further reduce the amount of particulates. Duringfiltration, in some embodiments, the pH is maintained between a pH ofabout 8.5 to about 12.0 (e.g., about 8.5 to about 9.0, about 9.0 toabout 10.0, about 9.0 to about 11.0, about 10.0 to about 11.0, about11.0 to about 12.0, about 9.5 to about 10.5, about 9.5 to about 11.5,about 10.5 to about 11.5, at 9.0, at 9.5, at 10.0, at 10.5, at 11.0, at11.5, or at 12.0). A cell lysate or clarified lysate can be filteredusing microfiltration, ultrafiltration, and/or diafiltration.Microfiltration can use a membrane with a pore size of about 0.2 μm toabout 2.0 μm (e.g., about 0.2 to about 0.3 μm, about 0.3 to about 0.5μm, about 0.5 to about 0.7 μm, about 0.7 to about 0.9 μm, about 0.9 toabout 1.1 μm, about 1.0 to about 1.2 μm, about 1.2 to about 1.4 μm,about 1.4 to about 1.6 μm, about 1.6 to about 1.8 μm, or about 1.8 toabout 2.0 μm). Ultrafiltration can use a membrane with a molecularweight cutoff of about 5 kDa to about 70 kDa (e.g., about 5 kDa to about10 kDa, about 10 kDa to about 30 kDa, or about 30 kDa to about 50 kDa,about 20 kDa to about 40 kDa, about 40 to about 60 kDa, or about 50 kDato about 70 kDa).

In some embodiments, a clarified lysate can be filtered to obtain afiltered lysate. In some embodiments, a filtration step can reduce theamount of particulates. During filtration, in some embodiments, the pHis maintained between a pH of about 8.5 to about 12.0 (e.g., about 8.5to about 9.0, about 9.0 to about 10.0, about 9.0 to about 11.0, about10.0 to about 11.0, about 11.0 to about 12.0, about 9.5 to about 10.5,about 9.5 to about 11.5, about 10.5 to about 11.5, at 9.0, at 9.5, at10.0, at 10.5, at 11.0, at 11.5, or at 12.0). A clarified lysate can befiltered using microfiltration, ultrafiltration, and/or diafiltration.Microfiltration can use a membrane with a pore size of about 0.2 μm toabout 2.0 μm (e.g., about 0.2 to about 0.3 μm, about 0.3 to about 0.5μm, about 0.5 to about 0.7 μm, about 0.7 to about 0.9 μm, about 0.9 toabout 1.1 μm, about 1.0 to about 1.2 μm, about 1.2 to about 1.4 μm,about 1.4 to about 1.6 μm, about 1.6 to about 1.8 μm, or about 1.8 toabout 2.0 μm). Ultrafiltration can use a membrane with a molecularweight cutoff of about 5 kDa to about 70 kDa (e.g., about 5 kDa to about10 kDa, about 10 kDa to about 30 kDa, about 30 kDa to about 50 kDa,about 20 to about 40 kDa, about 40 kDa to about 60 kDa, or about 50 toabout 70 kDa).

In some embodiments, a filtered lysate can be subjected to one or moreadditional filtration steps to obtain another filtered lysate. Afiltered lysate can be filtered using microfiltration, ultrafiltration,and/or diafiltration. Microfiltration can use a membrane with a poresize of about 0.2 μm to about 2.0 μm (e.g., about 0.2 to about 0.3 μm,about 0.3 to about 0.5 μm, about 0.5 to about 0.7 μm, about 0.7 to about0.9 μm, about 0.9 to about 1.1 μm, about 1.0 to about 1.2 μm, about 1.2to about 1.4 μm, about 1.4 to about 1.6 μm, about 1.6 to about 1.8 μm,or about 1.8 to about 2.0 μm). Ultrafiltration can use a membrane with amolecular weight cutoff of about 5 kDa to about 70 kDa (e.g., about 5kDa to about 10 kDa, about 10 kDa to about 30 kDa, about 30 kDa to about50 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, orabout 50 kDa to about 70 kDa). For example, a filtered lysate can befurther filtered by forcing the solution (e.g., using increased pressureor centrifugation) through a semi-permeable membrane having, forexample, a molecular weight cutoff of about 5 kDa to about 50 kDa (e.g.,about 5 kDa to about 10 kDa, about 10 kDa to about 30 kDa, or about 30kDa to about 50 kDa). In some embodiments, a filtered lysate can bediafiltered on a microfiltration membrane. In some embodiments, afiltered lysate can be diafiltered on an ultrafiltration membrane. Insome embodiments, filtered lysate (e.g., cell lysate or clarified lysatefiltered by microfiltration and/or ultrafiltration) can be concentratedto at least about 10% dry solids (e.g., at least about 15% or 20% drysolids) then diafiltered at constant volume for at least one diavolume(DV) (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 diavolumes).In some embodiments, filtered lysate (e.g., cell lysate or clarifiedlysate filtered by microfiltration and/or ultrafiltration) can bediluted (e.g., using water or an aqueous solution of salts or buffers,while maintaining the pH between about pH 8.5 and 12.0) to about 5% drysolids (e.g., about 6%, 7%, 8% or 9% dry solids) then diafiltered atconstant volume for at least one diavolume (e.g., at least 2, 3, 4, 5,6, 7, 8, 9, 10, 15, or 20 diavolumes). In some embodiments, filteredlysate (e.g., cell lysate or clarified lysate filtered bymicrofiltration and/or ultrafiltration) can be diluted (e.g., usingwater or an aqueous solution of salts or buffers, while maintaining thepH between about pH 8.5 and 12.0) to about 3% dry solids (e.g., about 2%or about 4% dry solids) then diafiltered to concentration the filteredlysate to about 15% dry solids (e.g., about 13%, 14%, 16%, or 17% drysolids). During the additional filtration step or steps, in someembodiments, the pH is maintained at a pH between about 8.5 to about12.0 (e.g., about 8.5 to about 9.0, about 9.0 to about 10.0, about 9.0to about 11.0, about 10.0 to about 11.0, about 11.0 to about 12.0, about9.5 to about 10.5, about 9.5 to about 11.5, about 10.5 to about 11.5, at9.0, at 9.5, at 10.0, at 10.5, at 11.0, at 11.5, or at 12.0).

A cell lysate can be concentrated (e.g., through filtering methods asdescribed for removing components smaller than the desired protein suchas ultrafiltration, optionally with diafiltration). During theconcentration, the pH can be maintained at a pH between about 8.5 toabout 12.0 (e.g., about 8.5 to about 9.0, about 9.0 to about 10.0, about9.0 to about 11.0, about 10.0 to about 11.0, about 11.0 to about 12.0,about 9.5 to about 10.5, about 9.5 to about 11.5, about 10.5 to about11.5, at 9.0, at 9.5, at 10.0, at 10.5, at 11.0, at 11.5, or at 12.0). Acell lysate can be concentrated to a protein content of about 2 mg/mL toabout 250 mg/mL (e.g., 10 mg/mL to 225 mg/mL, 15 mg/mL to 200 mg/mL, 25mg/mL to about 225 mg/mL, 50 mg/mL to 200 mg/mL, or 50 mg/mL to 150mg/mL). Concentration can occur concurrently with a filtration step.Concentration can occur separately from a filtration step.

A clarified lysate can be concentrated (e.g., through filtering methodsas described for removing components smaller than the desired proteinsuch as ultrafiltration, optionally with diafiltration). During theconcentration, the pH can be maintained at a pH between about 8.5 toabout 12.0 (e.g., about 8.5 to about 9.0, about 9.0 to about 10.0, about9.0 to about 11.0, about 10.0 to about 11.0, about 11.0 to about 12.0,about 9.5 to about 10.5, about 9.5 to about 11.5, about 10.5 to about11.5, at 9.0, at 9.5, at 10.0, at 10.5, at 11.0, at 11.5, or at 12.0). Aclarified lysate can be concentrated to a protein content of about 2mg/mL to about 250 mg/mL (e.g., 10 mg/mL to 225 mg/mL, 15 mg/mL to 200mg/mL, 25 mg/mL to about 225 mg/mL, 50 mg/mL to 200 mg/mL, or 50 mg/mLto 150 mg/mL). Concentration can occur concurrently with a filtrationstep. Concentration can occur separately from a filtration step.

A filtered lysate can be concentrated (e.g., through filtering methodsas described for removing components smaller than the desired proteinsuch as ultrafiltration, optionally with diafiltration). During theconcentration, the pH can be maintained at a pH between about 8.5 toabout 12.0 (e.g., about 8.5 to about 9.0, about 9.0 to about 10.0, about9.0 to about 11.0, about 10.0 to about 11.0, about 11.0 to about 12.0,about 9.5 to about 10.5, about 9.5 to about 11.5, about 10.5 to about11.5, at 9.0, at 9.5, at 10.0, at 10.5, at 11.0, at 11.5, or at 12.0). Afiltered lysate can be concentrated to a protein content of about 2mg/mL to about 250 mg/mL (e.g., 10 mg/mL to 225 mg/mL, 15 mg/mL to 200mg/mL, 25 mg/mL to about 225 mg/mL, 50 mg/mL to 200 mg/mL, or 50 mg/mLto 150 mg/mL). Concentration can occur concurrently with a filtrationstep. Concentration can occur separately from a filtration step.

In some embodiments, a protein composition, where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about12.0, can comprise at least 35% (e.g. at least 40%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 99%) on a dry weight basis of moleculeslarger than 500 Da (e.g., 1 kDa, 2 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, or50 kDa). A person of ordinary skill in the art can determine the totalamount of large molecules (e.g., molecules larger than 500 Da, 1 kDa, 2kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, or 50 kDa), or the amount of aparticular large molecule in a sample, using any of a variety of knownmethods, e.g., liquid chromatography-mass spectrometry (LCMS). A personof ordinary skill in the art can determine the total amount of smallmolecules (e.g., molecules smaller than 30 kDa, 20 kDa, 10 kDa, 5 kDa, 3kDa, 2 kDa, 1 kDa, or 500 Da), or the amount of a particular smallmolecule in a sample, using any of a variety of known methods, e.g.,liquid chromatography-mass spectrometry (LCMS). In some embodiments, areduction in small molecules (e.g., molecules smaller than 30 kDa, 20kDa, 10 kDa, 5 kDa, 3 kDa, 1 kDa, or 500 Da) can occur concurrently witha filtration step, e.g., diafiltration.

It will be appreciated that the choice of filter (e.g., membranematerial, pore size) and filtration method (e.g., microfiltration,ultrafiltration, or diafiltration) can affect or even dictate whether adesired component will be in the retentate or the filtrate of a givenfiltration step. For example, in some embodiments, if a large moleculeis a desired component, ultrafiltration can be selected as thefiltration method, and the desired component can be part of theretentate. In some embodiments, ultrafiltration can be combined withdiafiltration.

In some embodiments, steps in any of the methods described herein can beperformed independently at a pH of 8.5 to 12. For example, a lysing stepcan be performed at a pH of about 8.5 to about 9.0, about 9.0 to about10.0, about 9.0 to about 11.0, about 10.0 to about 11.0, about 11.0 toabout 12.0, about 9.5 to about 10.5, about 9.5 to about 11.5, about 10.5to about 11.5, at about 9.0, at about 9.5, at about 10.0, at about 10.5,at about 11.0, at about 11.5, or at about 12.0, while the clarifyingand/or filtering steps can each be independently performed at a pHdifferent from the pH of the lysing step, so long as the different pH ofthe clarifying and/or filtering steps is above 8.5. As another example,a clarifying step can be performed at a pH of about 8.5 to about 9.0,about 9.0 to about 10.0, about 9.0 to about 11.0, about 10.0 to about11.0, about 11.0 to about 12.0, about 9.5 to about 10.5, about 9.5 toabout 11.5, about 10.5 to about 11.5, at about 9.0, at about 9.5, atabout 10.0, at about 10.5, at about 11.0, at about 11.5, or at about12.0, while the lysing and/or filtering steps can each be independentlyperformed at a pH different from the pH of the clarifying step, so longas the different pH of the lysing and/or filtering steps is above 8.5.As another example, a filtering step can be performed at a pH of about8.5 to about 9.0, about 9.0 to about 10.0, about 9.0 to about 11.0,about 10.0 to about 11.0, about 11.0 to about 12.0, about 9.5 to about10.5, about 9.5 to about 11.5, about 10.5 to about 11.5, at about 9.0,at about 9.5, at about 10.0, at about 10.5, at about 11.0, at about11.5, or at about 12.0, while the lysing and/or clarifying steps caneach be independently performed at a pH different from the pH of thefiltering step, so long as the different pH of the lysing and/orclarifying steps is above 8.5.

In some embodiments, a protein composition, where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about12.0, can have a reduced the amount of or none of one or more smallmolecules that can contribute buffering capacity compared to purifiedprotein wherein the same purification step or steps were not carried outabout pH 8.5 and about 12.0. Ingredient buffering capacity cancontribute to pH drift, which can promote off-flavor development, aswell as potentially impacting product assembly and formulation. Forexample, a protein composition can have a buffering capacity of lessthan about 2.5 mmol NaOH per gram dry solids (e.g., less than about 2.4,2.3, 2.2, 2.1, 2.0, 1.9, 1.8. 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0,0.5, or 0.1 mmol NaOH per gram dry solids). In some embodiments,diafiltration (at, e.g., pH 9.310.3) can lower the buffering capacity(e.g., from about 3.6 mmol NaOH per gram dry solids) to less than about2.5 mmol NaOH per gram dry solids (e.g., less than about 2.4, 2.3, 2.2,2.1, 2.0, 1.9, 1.8. 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.5, or 0.1mmol NaOH per gram dry solids). The buffer capacity can be determined bypH titration of a 2% (w/v) suspension or solution, measuring the mmol ofNaOH that would be required to move the suspension or solution from pH3.0 to pH 12.0.

In some embodiments, a protein composition, where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about12.0, can form a gel with a higher storage modulus compared to purifiedprotein where the same purification step or steps were not carried outabout pH 8.5 and about 12.0. For example, in some embodiments, a gel canbe formed from a protein composition with a concentration of about 25 toabout 250 mg/mL (e.g., about 25 to about 50 mg/mL, about 25 to about 100mg/mL, about 25 to about 150 mg/mL, about 25 to about 200 mg/mL, about50 to about 250 mg/mL, about 100 to about 250 mg/mL, about 150 to about250 mg/mL, or about 200 to about 250 mg/mL) at a pH of about 7.0. Insome embodiments, a gel formed from a protein composition where one ormore purification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about 12.0at a 10% (w/v) suspension can have storage modulus greater than asimilar gel where the same purification step or steps were not carriedout about pH 8.5 and about 12.0. In some embodiments, the storagemodulus of a gel formed from a protein composition where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about 12.0at a 10% (w/v) suspension can have a storage modulus of at least about100 Pa at about 95° C. In some embodiments, the storage modulus of a gelat about 95° C. formed from a protein composition where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about 12.0at a 10% (w/v) suspension can have a storage modulus at least about10-fold (e.g., 15-fold or 20-fold) greater than the storage modulus of asimilar gel, where one or more purification steps (e.g., two or more,three or more, or all of the purification steps) were not carried outbetween about pH 8.5 and about 12.0. In some embodiments, the storagemodulus of a pasteurized (e.g., at 65° C. for 30 seconds) gel at about95° C. formed from a protein composition where one or more purificationsteps (e.g., two or more, three or more, or all of the purificationsteps) were carried out between about pH 8.5 and about 12.0 at a 10%(w/v) suspension can have a storage modulus at least about 2-fold (e.g.,3-fold, 4-fold, or 5-fold) greater than the storage modulus of a similargel, where one or more purification steps (e.g., two or more, three ormore, or all of the purification steps) were not carried out betweenabout pH 8.5 and about 12.0.

In some embodiments, a protein composition, where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about12.0, can have less (e.g., a smaller absolute amount or a smallerconcentration) of one or more small molecules that can contribute tooff-odors or off-flavors (e.g., cysteine, 1-Hexanol; 2-Butylfuran;2-methyl-2-Pentenal; 3-Octanone; Ethyl-Acetate; 2-Ethyl-Furan;2-pentyl-Furan; Pyrazine; 1-Decanol; Acetophenone; 1-Nonanol;2,5-Dimethyl-Pyrazine; Dodecanal; Benzeneacetaldehyde; Nonanal;Butyrolactone; Octanal; 2-Decanone; Hexanal; 2-Nonanone; Benzaldehyde;Heptanal; 2-Octanone; Furfural; 2-Heptanone; Pentanal) than a proteincomposition obtained by purification method in which one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were not carried out between about pH 8.5 and about12.0. In some embodiments, a protein composition, where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about12.0, can have a reduction of at least about 1.05-fold (e.g., at leastabout 2.0-fold, at least about 2.5-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, or at least about 10-fold) in oneor more small molecules that can contribute to off-odors or off-flavors(e.g., cysteine, 1-Hexanol; 2-Butylfuran; 2-methyl-2-Pentenal;3-Octanone; Ethyl-Acetate; 2-Ethyl-Furan; 2-pentyl-Furan; Pyrazine;1-Decanol; Acetophenone; 1-Nonanol; 2,5-Dimethyl-Pyrazine; Dodecanal;Benzeneacetaldehyde; Nonanal; Butyrolactone; Octanal; 2-Decanone;Hexanal; 2-Nonanone; Benzaldehyde; Heptanal; 2-Octanone; Furfural;2-Heptanone; Pentanal) compared to purified protein wherein the samepurification step or steps were not carried out about pH 8.5 and 12.0.In some embodiments, a fold-reduction can be calculated by dividing theamount of a small molecule in a protein composition where one or moresteps (e.g., two or more, three or more, or all of the purificationsteps) were not carried out between about pH 8.5 and about 12.0 by theamount of the same small molecule where the same purification step orsteps were carried out between about pH 8.5 and about 12.0. A person ofordinary skill in the art can determine the amount of a particular smallmolecule in a sample, using, e.g., GCMS.

In some embodiments, a protein composition, where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about12.0, includes one or more small molecules that contribute to off-odorsor off-flavors (e.g., cysteine, 1-Hexanol; 2-Butylfuran;2-methyl-2-Pentenal; 3-Octanone; Ethyl-Acetate; 2-Ethyl-Furan;2-pentyl-Furan; Pyrazine; 1-Decanol; Acetophenone; 1-Nonanol;2,5-Dimethyl-Pyrazine; Dodecanal; Benzeneacetaldehyde; Nonanal;Butyrolactone; Octanal; 2-Decanone; Hexanal; 2-Nonanone; Benzaldehyde;Heptanal; 2-Octanone; Furfural; 2-Heptanone; Pentanal) in an amountbelow a level detectable by a panelist. A person of ordinary skill inthe art can determine the amount of a particular small molecule in asample, using, e.g., GCMS.

In some embodiments, a protein composition, where one or morepurification steps (e.g., two or more, three or more, or all of thepurification steps) were carried out between about pH 8.5 and about12.0, has no detectable level of one or more small molecules thatcontribute to off-odors or off-flavors (e.g., cysteine, 1-Hexanol;2-Butylfuran; 2-methyl-2-Pentenal; 3-Octanone; Ethyl-Acetate;2-Ethyl-Furan; 2-pentyl-Furan; Pyrazine; 1-Decanol; Acetophenone;1-Nonanol; 2,5-Dimethyl-Pyrazine; Dodecanal; Benzeneacetaldehyde;Nonanal; Butyrolactone; Octanal; 2-Decanone; Hexanal; 2-Nonanone;Benzaldehyde; Heptanal; 2-Octanone; Furfural; 2-Heptanone; Pentanal).

A protein composition also can be pasteurized. For example, a proteincomposition can be pasteurized using heat treatment, high temperatureshort time pasteurization, pulsed electric field, high pressurepasteurization, UV irradiation, gamma irradiation, or microfiltration.In some embodiments, one or more antimicrobials (e.g., polylysine) canbe added during or after pasteurization.

In some embodiments, a protein composition, whether pasteurized or not,can be dried, e.g., spray dried or freeze dried or the like under mildconditions to ensure that the protein is not denatured.

In some embodiments, at least about 50% (e.g., at least about 60%, 70%,80%, or 90%) of polypeptides in a protein composition fall between about10 kDa and about 200 kDa when measured by reducing and denaturing gelelectrophoresis (e.g., SDS-PAGE) and densitometry after detection in amanner common to the art (e.g., Coomassie Brilliant Blue G-250,Coomassie Brilliant Blue R-250, or silver nitrate).

In some embodiments, one or more proteins in a protein composition, asdescribed herein is functional (as described above). In someembodiments, a protein composition can be used in a variety of foodproducts, including, for example, protein supplements (e.g., proteinpowders), meal replacements, or baked goods, or to replace all or aportion of an animal protein (e.g., from cows, pigs, poultry, lamb, orfish) in a food product that mimics an animal derived food product(e.g., a cheese replica, an egg replica or meat replica such as a groundmeat replica).

In some embodiments, a protein composition can include one or moreproteins selected from the group consisting of AOX1 and AOX2.

In some embodiments, a protein composition can include one or moreproteins, identified by GI number, selected from the group consistingof: 254568470, 254567507, 254570367, 254568544, 254573764, 254566601,254566257, 254567798, 254570575, 254571387, 254571425, 254568572,254571679, 254569858, 254573010, 254564691, 254571699, 254572585,254566127, 254570667, 254572870, 254573696, 254565205, 254569186,254572668, 254571899, 254569222, 254572359, 254573464, 254572163,254570957, 254573014, 254570673, 254566987, 254567581, 254564747,254568562, 254566731, 254565437, 254564519, 254571763, 254566729,254569372, 254571423, 254565451, 254565973, 254573008, 254574310,254564587, 254568946, 254569478, 254566861, 254565513, 254572906,254572796, 254573092, 254567233, 254565959, 254570383, 254570885,254565519, 254574530, 254573558, 254569654, 254573466, 254571991,254568780, 254565859, 254568564, 254570088, 254564995, 254573142,254571407, 254569976, 254570771, 254565455, 254565551, 254574244,254567716, 254572952, 254568654, 254570661, 254566481, 254565045,254567189, 254570098, 254566883, 254569212, 254574528, 254565655,254568196, 254572782, 254570305, 254572856, 254568894, 254564809,254569780, 254565263, 254567287, 254567754, 254565279, 254567471,254564667, 254568442, 254571893, 254573996, 254572005, 254572033,254567027, 254569734, 254566559, 254570993, 254566611, 254566089,254567714, 254571057, 254573908, 254572676, 254570100, 254567055,254565475, 254567834, 254567872, 254566327, 254574366, 254568036,254567738, 254565307, 254570727, 254566301, 254565783, 254567173,254568492, 254565493, 254571145, 254565699, 254567029, 254573198,254568642, 254568302, 254574036, 254569716, 254571157, 254571485,254567788, 254573482, 254566631, 254571293, 254569576, 254572962,254565129, 254569094, 254570112, 254566447, 254570515, 254568944,254573180, 254573098, 254570969, 254569500, 254564507, 254569714,254570072, 254573324, 254568444, 254569156, 254568334, 254568304,254566885, 254571359, 254574464, 254569852, 254569144, 254573818,254573050, 254572992, 254571587, 254573470, 254565991, 254566975,254570022, 254569102, 254574442, 254569298, 254572501, 254569162,254565713, 254565211, 254572377, 254571575, 254574030, 254572872,254565431, 254569400, 254572599, 254565725, 254569842, 254570271,254573426, 254574502, 254569568, 254572822, 254568950, 254572257,254569700, 254573726, 254574210, 254567569, 254569896, 254571915,254568132, 254567191, 254566971, 254567243, 254566843, 254568386,254571131, 254572628, 254567025, 254569344, 254571043, 254572958,254566489, 254566607, 254569916, 254571179, 254569898, 254567938,254566619, 254566141, 254572159, 254573164, 254573328, 254570166,254565545, 254570535, 254564705, 254570393, 254566769, 254565063,254567203, 254573714, 254571903, 254571639, 254564825, 254569512,254569682, 254574296, 254574080, 254564849, 254570719, 254568186,254572093, 254573284, 254571919, 254570821, 254565769, 254573468,254571883, 254570633, 254570315, 254570527, 254567583, 254573420,254569866, 254569290, 254569438, 254574316, 254566267, 254570897,254569696, 254566847, 254572974, 254569386, 254568682, 254565735,254574242, 254568876, 254566225, 254566479, 254569106, 254566881,254569226, 254565085, 254569736, 254572157, 254565157, 254573986,254569320, 254570679, 254572211, 254566063, 254568616, 254564917,254564915, 254568842, 254573376, 254566487, 254565875, 254568412,254564663, 254565961, 254569890, 254566293, 254568216, 254572836,254570523, 254568506, 254572347, 254567662, 254567720, 254574020,254571733, 254571747, 254569894, 254571377, 254566013, 254569558,254565617, 254566191, 254571955, 254565721, 254567996, 254566897,254574140, 254571035, 254570359, 254566893, 254568298, 254566101,254565989, 254568566, 254571457, 254571393, 254571161, 254564537,254571649, 254570979, 254566595, 254566417, 254569072, 254570669,254569122, 254567253, 254573448, 254571343, 254572333, 254566649,254571369, 254573496, 254572133, 254573886, 254569410, 254570411,254565705, 254573462, 254568908, 254572495, 254569552, 254566933,254568102, 254569702, 254569846, 254574136, 254569390, 254567273,254572371, 254569354, 254572053, 254568006, 254565653, 254570487,254573510, 254564923, 254568606, 254572503, 254569166, 254572145,254572531, 254568464, 254570723, 254570485, 254567349, 254565049,254574132, 254564979, 254564629, 254572309, 254565163, 254573160,254573452, 254565165, 254573834, 254574102, 254568996, 254573756,254572015, 254568992, 254572535, 254566143, 254572321, 254568056,254573266, 254566351, 254571007, 254571463, 254570629, 254573948,254572884, 254567375, 254567467, 254567928, 254572307, 254567700,254571783, 254573676, 254567900, 254568714, 254564921, 254564599,254573602, 254573662, 254570521, 254572814, 254571619, 254566321,254568896, 254566743, 254572553, 254565607, 254565035, 254565403,254573760, 254566097, 254564603, 254572956, 254564565, 254572834,254566317, 254564665, 254573056, 254568582, 254564717, 254572724,254565955, 254568718, 254573174, 254566999, 254569742, 254573508,254565095, 254569494, 254573046, 254568148, 254574028, 254569828,254566605, 254569010, 254568244, 254564675, 254569952, 254574158,254567143, 254566779, 254574118, 254573866, 254570090, 254569928,254573178, 254574370, 254569876, 254571543, 254570799, 254569038,254570373, 254567774, 254571247, 254574144, 254571313, 254570649,254565795, 254572920, 254568600, 254571879, 254567231, 254567553,254569782, 254566517, 254566423, 254573664, 254565207, 254566497,254566087, 254571791, 254568702, 254569082, 254569470, 254565589,254570561, and 254574100. A GI number can be searched in in the PubMedprotein database found at ncbi.nlm.nih.gov/protein, e.g., to retrievethe name and/or sequence of the corresponding protein.

In some embodiments, a protein composition can include one or moreproteins selected from a Pfam family selected from the group consistingof Pf00044, Pf02800, Pf02826, Pf00009, Pf03143, Pf03144, Pf00113,Pf03952, Pf00107, Pf08240, Pf00012, Pf06723, Pf00162, Pf00183, Pf02518,Pf00009, Pf00679, Pf03144, Pf03764, Pf00205, Pf02775, Pf02776, Pf00006,Pf00306, Pf02874, Pf01249, Pf00240, Pf01020, Pf11976, Pf00240, Pf01599,Pf11976, Pf00006, Pf00306, Pf02874, Pf00153, Pf00189, Pf07650, Pf00012,Pf06723, Pf01929, Pf00400, Pf00012, Pf06723, Pf00297, Pf01015, Pf01116,Pf00224, Pf02887, Pf03328, Pf00005, Pf03193, Pf00300, Pf00416, Pf01287,Pf01294, Pf00121, Pf00012, Pf06723, Pf01251, Pf00022, Pf00318, Pf00327,Pf00238, Pf00411, Pf01090, Pf00687, Pf00573, Pf00177, Pf00828, Pf01459,Pf00270, Pf00271, Pf00298, Pf03946, Pf01201, Pf00572, Pf00276, Pf00428,Pf00466, Pf00827, Pf00923, Pf01781, Pf01248, Pf00118, Pf00043, Pf00647,Pf00900, Pf01479, Pf08071, Pf00333, Pf03719, Pf01450, Pf07991, Pf01280,Pf01780, Pf00181, Pf03947, Pf03501, Pf00312, Pf08069, Pf00338, Pf00380,Pf00132, Pf00483, Pf12804, Pf00366, Pf00347, Pf00410, Pf00334, Pf00160,Pf01248, Pf00237, Pf00393, Pf03446, Pf00213, Pf00347, Pf00080, Pf01248,Pf01198, Pf00838, Pf01200, Pf00122, Pf00690, Pf00702, Pf08282, Pf12710,Pf00252, Pf00163, Pf01479, Pf00833, Pf01092, Pf01667, Pf00107, Pf08240,Pf01092, Pf01775, Pf01159, Pf01667, Pf01849, Pf00125, Pf02969, Pf00675,Pf05193, Pf00153, Pf00244, Pf00208, Pf02812, Pf01158, Pf00330, Pf00694,Pf00125, Pf00808, Pf02284, Pf00281, Pf00673, Pf00438, Pf02772, Pf02773,Pf00670, Pf02826, Pf05221, Pf00428, Pf00578, Pf08534, Pf10417, Pf01247,Pf02953, Pf09598, Pf04969, Pf01246, Pf00202, Pf01212, Pf00349, Pf03727,Pf01776, Pf03332, Pf08282, Pf00253, Pf00155, Pf00012, Pf06723, Pf01717,Pf08267, Pf00166, Pf00085, Pf00056, Pf02866, Pf00076, Pf00658, Pf00285,Pf00406, Pf05191, Pf00456, Pf02779, Pf02780, Pf00861, Pf00349, Pf03727,Pf00025, Pf00071, Pf01926, Pf04670, Pf08477, Pf05873, Pf00342, Pf00831,Pf00203, Pf01282, Pf00515, Pf07719, Pf01215, Pf01159, Pf05405, Pf00180,Pf02297, Pf00108, Pf00109, Pf02803, Pf01488, Pf03435, Pf05368, Pf02953,Pf00076, Pf00012, Pf06723, Pf00828, Pf00070, Pf01262, Pf02852, Pf07992,Pf12831, Pf00793, Pf11022, Pf00389, Pf00670, Pf02826, Pf05221, Pf00085,Pf02114, Pf01063, Pf01209, Pf02353, Pf08241, Pf08242, Pf08498, Pf12847,Pf03297, Pf00719, Pf00254, Pf00226, Pf00684, Pf01556, Pf00164, Pf00125,Pf00627, Pf01849, Pf00736, Pf01283, Pf01157, Pf00009, Pf03143, Pf03144,Pf05680, Pf00180, Pf04911, Pf00180, Pf01926, Pf06071, Pf00270, Pf00271,Pf00310, Pf00733, Pf00266, Pf01243, Pf10590, Pf00231, Pf00025, Pf00071,Pf00503, Pf01926, Pf04670, Pf08477, Pf09439, Pf01588, Pf01253, Pf00675,Pf02136, Pf00036, Pf00006, Pf00306, Pf02874, Pf00410, Pf01655, Pf00085,Pf01546, Pf00270, Pf00271, Pf04851, Pf00025, Pf00071, Pf00503, Pf01926,Pf08477, Pf09439, Pf00254, Pf00006, Pf00306, Pf02874, Pf04568, Pf00956,Pf00180, Pf00133, Pf01406, Pf08264, Pf09334, Pf00004, Pf00910, Pf01078,Pf02359, Pf02933, Pf05496, Pf05673, Pf06068, Pf07724, Pf07728, Pf00676,Pf12718, Pf01808, Pf02142, Pf02167, Pf11578, Pf00091, Pf03953, Pf01873,Pf02020, Pf00702, Pf01030, Pf00226, Pf00083, Pf07690, Pf00587, Pf02403,Pf02779, Pf02780, Pf01798, Pf08060, Pf08156, Pf00365, Pf00106, Pf01073,Pf01370, Pf07993, Pf08659, Pf00034, Pf00155, Pf01041, Pf01053, Pf00549,Pf01071, Pf08442, Pf00501, Pf11930, Pf03114, Pf10455, Pf01199, Pf00106,Pf00109, Pf01648, Pf02801, Pf00291, Pf00571, Pf01118, Pf02774, Pf08718,Pf01154, Pf08540, Pf00070, Pf07992, Pf00081, Pf02777, Pf00152, Pf01336,Pf01798, Pf08060, Pf08156, Pf10642, Pf00289, Pf00364, Pf00682, Pf02436,Pf02785, Pf02786, Pf07478, Pf00180, Pf09229, Pf01704, Pf00076, Pf00887,Pf00698, Pf01575, Pf03060, Pf08354, Pf05047, Pf00155, Pf01347, Pf00549,Pf02629, Pf00076, Pf08662, Pf00018, Pf00241, Pf07653, Pf00070, Pf01946,Pf07992, Pf01269, Pf00133, Pf08264, Pf09334, Pf00117, Pf00958, Pf02540,Pf03054, Pf06508, Pf07722, Pf02550, Pf00479, Pf02781, Pf00005, Pf03193,Pf12848, Pf00155, Pf00266, Pf01041, Pf01053, Pf01212, Pf02347, Pf03841,Pf00009, Pf03144, Pf09173, Pf00118, Pf01907, Pf00155, Pf00464, Pf04718,Pf00733, Pf00764, Pf03054, Pf06508, Pf10791, Pf00926, Pf04669, Pf01459,Pf00294, Pf01192, Pf04281, Pf00638, Pf01873, Pf01399, Pf00587, Pf02824,Pf03129, Pf07973, Pf01920, Pf00743, Pf07992, Pf00255, Pf00578, Pf08534,Pf03134, Pf02271, Pf00120, Pf03951, Pf00310, Pf01380, Pf09280, Pf01634,Pf08029, Pf09084, Pf00682, Pf00501, Pf00176, Pf00270, Pf00271, Pf00437,Pf00625, Pf00910, Pf05729, Pf00198, Pf00364, Pf02817, Pf00171, Pf00705,Pf02144, Pf02747, Pf01652, Pf00241, Pf00171, Pf03198, Pf07983, Pf03198,Pf07983, Pf04627, Pf01042, Pf00152, Pf01336, Pf00682, Pf08502, Pf01912,Pf00578, Pf08534, Pf10417, Pf00226, Pf01556, Pf01399, Pf09440, Pf00262,Pf00118, Pf00750, Pf03485, Pf05746, Pf00111, Pf00384, Pf09326, Pf10588,Pf01248, Pf00085, Pf00462, Pf00676, Pf02779, Pf00009, Pf00071, Pf02421,Pf08477, Pf05091, Pf00133, Pf08264, Pf09334, Pf10458, Pf01472, Pf01509,Pf08068, Pf00118, Pf01111, Pf00160, Pf00152, Pf01336, Pf00899, Pf02134,Pf09358, Pf10585, Pf00682, Pf04111, Pf00175, Pf00970, Pf08030, Pf03435,Pf00575, Pf07541, Pf00332, Pf01257, Pf00742, Pf03447, Pf01262, Pf05222,Pf00832, Pf12710, Pf01266, Pf01411, Pf02272, Pf07973, Pf00013, Pf01991,Pf06505, Pf00587, Pf03129, Pf01398, Pf11976, Pf09796, Pf00025, Pf00071,Pf04670, Pf08477, Pf01176, Pf00043, Pf00749, Pf03950, Pf02374, Pf06244,Pf02939, Pf00160, Pf00515, Pf07719, Pf00793, Pf00709, Pf00235, Pf02115,Pf00881, Pf11885, Pf02823, Pf00291, Pf10276, Pf00004, Pf00158, Pf06414,Pf07724, Pf07726, Pf07728, Pf01433, Pf00155, Pf00076, Pf00118, Pf01194,Pf00317, Pf02867, Pf03477, Pf03483, Pf03484, Pf00076, Pf12353, Pf02453,Pf05262, Pf00578, Pf08534, Pf01238, Pf01564, Pf01218, Pf00227, Pf10584,Pf00240, Pf00627, Pf11976, Pf00153, Pf00009, Pf00025, Pf00071, Pf04670,Pf08477, Pf09439, Pf00350, Pf01031, Pf02212, Pf00535, Pf00890, Pf02910,Pf00583, Pf00403, Pf12223, Pf02854, Pf12152, Pf00152, Pf00587, Pf01409,Pf00004, Pf01057, Pf01078, Pf06068, Pf07724, Pf07726, Pf07728, Pf00155,Pf00464, Pf01381, Pf08523, Pf12844, Pf00156, Pf00735, Pf01926, Pf03193,Pf00004, Pf01057, Pf01078, Pf05673, Pf06068, Pf07726, Pf07728, Pf00290,Pf00291, Pf01208, Pf01466, Pf03931, Pf08327, Pf09229, Pf00107, Pf08240,Pf03223, Pf12757, Pf09731, Pf00557, Pf01753, Pf02936, Pf01793, Pf00155,Pf00202, Pf00155, Pf00687, Pf00091, Pf03953, Pf08597, Pf00118, Pf00586,Pf01071, Pf02222, Pf02655, Pf02769, Pf02843, Pf02844, Pf08442, Pf00118,Pf00343, Pf03130, Pf00332, Pf00270, Pf00271, Pf00004, Pf05496, Pf06068,Pf06414, Pf01145, Pf00579, Pf00266, Pf01212, Pf01965, Pf00815, Pf01502,Pf01503, Pf00149, Pf00542, Pf00156, Pf03098, Pf00400, Pf03604, Pf00248,Pf00365, Pf04145, Pf00400, Pf00329, Pf01086, Pf00004, Pf00158, Pf02861,Pf07724, Pf07728, Pf10431, Pf00205, Pf02775, Pf02776, Pf00043, Pf02798,Pf01546, Pf00227, Pf10584, Pf00156, Pf00310, Pf00118, Pf01012, Pf01145,Pf00481, Pf00248, Pf00206, Pf10397, Pf01602, Pf08752, Pf00227, Pf10584,Pf00491, Pf00300, Pf05739, Pf00004, Pf03796, Pf06068, Pf00107, Pf08240,Pf00298, Pf03946, Pf01399, Pf04135, Pf00637, Pf03463, Pf03464, Pf03465,Pf02330, Pf08662, Pf01512, Pf10531, Pf10589, Pf10785, Pf12853, Pf00735,Pf03193, Pf04548, Pf00635, Pf00650, Pf03765, Pf02656, Pf04758, Pf00731,Pf02222, Pf02655, Pf07478, Pf00118, Pf00275, Pf00465, Pf01202, Pf01487,Pf01488, Pf01761, Pf08501, Pf07957, Pf04280, Pf01399, Pf08375, Pf05383,Pf00076, Pf05383, Pf00636, Pf01641, Pf03678, Pf00125, Pf00364, Pf02817,Pf00462, Pf00227, Pf10584, Pf00291, Pf00585, Pf01263, Pf01399, Pf05470,Pf00459, Pf01576, Pf05911, Pf12128, Pf12757, Pf01398, Pf00009, Pf01926,Pf03029, Pf08597, Pf11987, Pf00390, and Pf03949.

In some embodiments, a protein composition can include one or moreproteins selected from the group consisting of Adh1, Adh2, Cit2, Eno1,Eno2, Fba1, Hxk1, Hxk2, Icl1, Pdb1, Pdc1, Pfk1, Pgi1, Pgk1, Pyc1, Tal1,Tdh2, Tdh3, Tpi1, Efb1, Eft1, Eft2, Prt1, Rpa0, Tif1,2, Yef3, Hsc82,Hsp60, Hsp82, Hsp104, Kar2, Ssa1, Ssa2, Ssb1, Ssb2, Ssc1, Sse1, Sti1,Ade1, Ade3, Ade5,7, Arg4, Gdh1, Gln1, His4, Ilv5, Lys9, Met6, Pro2,Ser1, Trp5, Act1, Adk1, Ald6, Atp2, Bmh1, Bmh2, Cdc19, Cdc48, Cdc60,Erg20, Gpp1, Gsp1, Ipp1, Lcb1, Mol1, Pab1, Pma1, Psa1, Rnr4, Sam1, Sam2,Sod1, Uba1, YKL056, YLR109, and YMR116.

In some embodiments, a protein composition can include one or moreproteins selected from the group consisting of cspB, cspD, rp1L, rp1U,hag, rpsN, rp1D, and yweA.

In some embodiments, a protein composition can include one or moreproteins selected from the group consisting of hup, ptsH, dpsA, tuf,gapB, rp1X, malE, and yhjA.

In some embodiments, a protein composition can include one or moreproteins selected from the group consisting of uspA, tufa, yqiA, rp1E,1pp, rp1Y, gatB, and rp1L.

In some embodiments, a protein composition can include one or moreproteins, identified by SwissProt accession number, selected from thegroup consisting of P00575, P06958, P00577, P02996, P04475, P02349,P06139, P09373, P02990, P17547, P22257, P06959, P06977, P11665, P14178,P02997, P00957, P00350, P07813, P23843, P00956, P08324, P08839, P02995,P07650, P03815, P09831, P05055, P00882, P00961, P07118, P09743, P10413,P60422, P02934, P00391, P30148, P04079, P36683, P12283, P06711, P00477,P02351, P0A8N3, P08177, P39184, P02384, P02354, P00968, P06981, P0A6T1,P07395, P08200, P27302, P62593, P03002, P09097, P11604, P16659, P15639,P00824, P02359, P00574, P60438, P00962, P62399, P15254, P07015, P26427,P23721, P00959, P00864, P02352, P03003, P39171, P62707, P39170, P15046,P02392, P17242, P00452, P14926, P00561, P25739, P00490, P02356, P76116,P04805, P00822, P00509, P23304, P07651, P32132, P30136, P17169, P21889,P08398, P61175, P00955, P08202, P08936, P29132, P06996, P04790, P04825,P03948, P02418, P09156, P15288, P32176, P00448, P33136, P08328, P02390,P17963, P22783, P02925, P60723, P02408, P08859, P09169, P13029, P16174,P25716, P04384, P21202, P02999, P30850, P33602, P35340, P05082, P08837,P37797, P02410, P22259, P07459, P10408, P22523, P02358, P09376, P45523,P00353, P06612, P33195, P08312, P24182, P12758, P17579, P00579, P07460,P61889, P25715, P60624, P09625, P23861, P22992, P33633, P07012, P17288,P27430, P60240, P02413, P37689, P32168, P00951, P08330, P18391, P21155,P07016, P13519, P21170, P06998, P02369, P02928, P02361, P11454, P06982,P02420, P77241, P31120, P36546, Q46829, P00954, P39172, P02426, P31216,P45577, P60906, P06138, P19673, P09372, P21513, P10177, P09151, P00891,P60785, P76177, P36938, P61517, P28691, P11457, P02428, P02419, P02416,P46837, P33599, P37747, P00913, P02931, P09546, P06971, P11096, P09157,P00934, P23480, P00960, P77482, P21346, P77349, P02364, P25665, P33138,P02375, P11875, P37095, P39435, P27827, P00479, P27248, P21599, P30867,P02363, P0A8N5, P22106, P04425, P37901, P02411, P02409, P39174, P02432,P39173, P10377, P25532, P31554, P02378, P24249, P30859, P03020, P37191,P37759, P23839, P77645, P33998, P76268, P02930, P24199, P02342, P14177,P07672, P23847, P63020, P08374, P08204, P27298, P02366, P24991, P05380,P17315, P21167, P21165, P23869, P31224, P17114, P76558, P15877, P19935,P07176, P61714, P10378, P24237, P60651, P77395, P17117, P24167, P06715,P37744, P02421, P25553, P24171, P05053, P03026, P08957, P00393, P02430,P27290, P02370, P04287, P23851, P00963, P17577, P39179, P10344, P09832,P07638, P76344, P00946, P38489, P45955, P05838, P75780, P23844, P31979,P00886, P11285, P07912, P25520, P00907, P02422, P18197, P26616, P07671,P52697, P02341, P39311, P33221, P39168, P00837, P22767, P19675, P05793,P62620, P02373, P45390, P00582, P77146, P30958, P24233, P05640, P16921,P07006, P30017, P00496, P31223, P36541, P02372, P76372, P31550, P39182,P11668, P21499, P77718, P10444, P19245, P02371, P08178, P18843, P45578,P21888, P22786, P02367, P23893, P23882, P11648, P51001, P02379, P10121,P05020, P24231, P02427, P60757, P15002, P31663, P19494, P08193, P37051,P02424, P13036, P02429, P00274, P15640, P02414, P36997, P0A6A6, P07004,P02435, P32164, P77310, P27252, P13652, P52643, P02436, P15277, P77804,P31057, P30139, P11028, P80063, P21774, P08622, P04951, P02374, P15716,P03017, P37648, P00923, P04422, P11557, P16456, P07906, P09159, P15048,Q46856, P39377, P14374, P06128, P29464, P60716, P00453, P37192, P76492,P45464, P23887, P00495, P45803, P33363, P30849, P04036, P18274, P28635,P77774, P46853, P25521, P14175, P36658, P39287, P78258, P77348, P30746,P29209, P24186, P26650, P23865, P05459, P15040, P30125, P25528, P30856,P36996, P08186, P02901, P33398, P39831, P18400, P23836, P20752, P29015,P04693, P00859, P02339, P36979, P60560, P0A6T3, P23858, P05825, P09424,P00831, P39330, P15047, P76153, P23853, P04816, P33598, P02998, P27251,P25714, P21892, P37754, P37329, P28909, P37187, P21590, P28302, P09029,P02937, P55741, P25662, P15039, P23863, P27851, P00370, P23932, P02905,P07019, P76002, P75876, P37688, P03025, P78083, P52065, P39406, P77258,P30744, P61316, P77254, P24253, P39811, P07005, P11026, P40874, P36540,P00478, P02437, P75789, P36766, P03844, P37010, P26428, P37190, P24250,P77438, P06984, P27434, P37749, P10384, Q57261, P15770, P00501, P24247,P77734, P12996, P42641, Q47130, P60546, P06129, P24223, P75838, P43675,P28694, P75902, P09375, P76403, P76658, P25529, P25516, P15034, P09200,P10902, P06995, P00547, P29210, P00583, P06613, P0A6W9, P75802, P28904,P31803, P25661, P27511, P30126, P00470, P30177, P17952, P10443, P37665,P36671, P76351, P36950, P09028, P00832, P06999, P23331, P07862, P09170,P40120, P80449, P77486, P14189, P06992, P05054, P75864, P09158, P61949,P62768, P07024, P23929, P75844, P07913, P37666, P00373, P04982, P03842,P76536, P07014, P13035, P36559, P76055, P36539, P09030, P21504, P36767,P39169, P08756, P42617, P32661, P37765, P23827, P04381, P52054, P20082,P09147, P06988, P76367, P46143, P05797, P77150, P06983, P25397, P18133,P75790, P16244, P08956, P37634, P43329, P24229, P06968, P75743, P28242,P18783, P27291, P30138, P45467, P06975, P46885, P39199, P10440, P25745,P40681, P25437, P33648, P37760, P75805, P00894, P77695, P00510, P31222,P09830, P31059, P05826, P76258, P76569, P18198, P46880, P30977, P07001,P45391, P13024, P13009, P33635, P24176, P31142, P17112, P60752, Q93K97,P11458, P08331, P37620, Q46828, P13000, P26615, P33644, P02917, P33918,P25888, P19934, P77338, P13685, P28225, P09997, P40718, P27828, P23830,P08188, P03812, P52647, P37667, Q46918, P00482, P18401, P32052, P03841,P62623, P46889, P27190, P37026, P11666, P39164, P46130, P30860, P37188,P76576, P33921, P31221, P37687, P12281, P76506, P25894, P00893, P03843,P25663, P45571, P77552, P52635, P30137, P76494, P39099, P24201, P20083,P46132, P76034, P39315, P09323, P37163, P07011, P31465, P39321, P05194,P77225, P32691, P37902, P09371, P77484, P23486, P39290, P76008, P32165,P19677, P76270, P45396, P75950, P77247, P75915, P32175, P05828, Q59384,P27306, P05848, P45748, P31133, P39396, P06986, P05796, P10740, P33570,P46473, P28690, P32130, P17993, P39177, P31664, P23911, P43671, P30848,P21338, Q46920, P77392, P61320, P23003, P39202, P45533, P15042, P30010,P02943, P32126, P26282, P46186, P38521, P09053, P00642, P25907, P00562,P17580, P09152, P17994, P76277, P76504, P75947, P37096, P37066, P52049,P02914, Q46933, P22333, P29217, P07020, P15298, P03807, P37631, P33597,P37347, P08367, P07002, P28304, P52061, P39356, P37308, Q46871, P15302,P00363, P75914, Q46948, P22563, P37345, P11056, P05791, P33601, P28633,P08373, P42550, P17113, P77202, P31218, P37175, P32157, P29679, P24178,P29680, P75736, P22188, P45389, P76290, P55139, P21645, P17448, P55253,P37440, P36564, P24245, P76370, P36995, P45799, P33636, P32105, Q46837,P23909, P78067, P21169, P08390, P30748, P16680, P36680, P41407, P76110,P23930, P28692, P16095, P03018, P15977, P21829, P09148, P05021, P23483,P31658, P45847, P39286, P46860, P40191, P37350, 065938, P32680, P12008,P27303, P03817, P46930, P21507, P77499, P76550, P52083, P37346, P33016,P09551, P24251, P25519, P11721, P27292, P00928, P17445, P43672, P33650,P24218, P07604, P39335, P28637, P29745, O69415, P71295, P11603, P76272,P32099, P77455, P45426, P15484, P15028, P08323, P00550, P02918, P30870,P76503, P24183, P36672, P23874, P03818, P02978, P33349, P75783, P33916,Q46863, P27848, P23199, P25533, P36768, P19641, P76423, P18393, P27841,P03019, P45580, P08660, P61887, P39401, P23894, P23884, P33643, P19674,P00811, P08179, P40717, P07085, P18390, P75849, P33031, P37189, P39323,P22938, P10346, P37647, P23089, P76187, P24285, P75823, P37745, P76426,P28861, Q46872, P75958, P02924, P60340, Q47622, P32174, P03033, P32703,P43781, P75949, P15050, P37349, P76316, P25738, P11288, P24203, P10957,P76015, P08203, P37354, P27838, P17109, P34086, P76141, P31220, P27550,P51024, P46131, P28248, P31680, P37606, Q46893, Q46868, P08244, P16528,P20099, P39903, P07003, P77293, P45756, P24213, P21516, P37692, P75745,P32695, P37194, P27829, P76495, P45529, P52124, P75968, P00844, P11027,P52084, P33220, P33362, P77605, P22255, P00926, P26648, P30854, P33129,P32050, P15272, P06149, P32177, P75957, P11349, P77674, P32678, P76036,P30858, P12610, P23870, P36879, P37904, P39347, P18196, P17443, P36929,P31546, P26646, P03004, P31828, P05792, P30178, P33353, P29011, P30855,Q00191, P77561, P76496, P77252, P32721, P08338, P18775, P37330, P33940,P76422, P07676, Q46841, P45535, P30846, P06964, P23282, P39833, P33226,P76017, P52052, P45471, P03021, P23917, P11880, P60472, P36565, P77624,P07762, P28689, P06716, P22256, P45802, Q52280, P75913, P46474, P19635,P09391, P15038, P22997, Q57154, P08577, P75874, P76146, P24181, P22763,P27850, P77239, P37005, Q46814, P37626, P77562, P39835, P76256, P77500,P24205, P06712, P09454, P11257, P75793, P42908, P31475, P76014. ASwissProt accession number can be searched in in the UniProt proteindatabase found at uniprot.org, e.g., to retrieve the name and/orsequence of the corresponding protein.

In some embodiments, a protein composition can include a heme-containingprotein. In some embodiments, a protein composition can include one ormore proteins selected from the group consisting of an androglobin, acytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, aleghemoglobin, a flavohemoglobin, Hell's gate globin I, a myoglobin, anerythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, acyanoglobin, a cytoglobin, a histoglobin, a neuroglobins, achlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated2/2 globin, a hemoglobin 3 (e.g., Glb3), a cytochrome, or a peroxidase.

In some embodiments, a protein composition can include carbohydratepolymers (e.g., beta-glucan, glycogen, xanthan, xylinan, gellan,curdlan, agarose, dextran, pullulan, teichoic acids, peptidoglycan(e.g., murein) or nucleic acid polymers (e.g., ribosomal RNA (rRNA),transfer RNA (tRNA), messenger RNA (mRNA), or genomic DNA), or otherbiopolymers.

In some embodiments, a protein composition can include a heterologouslyexpressed protein. In some embodiments, a heterologously expressedprotein may be from a species different from the host cell, for examplefrom a eukaryote, an animal, a plant, an algae, a thermophile, a yeast,a bacteria, a protist or an archea. In some embodiments, aheterologously expressed protein can be any of the proteins describedherein. In some embodiments, a heterologously expressed protein can be aheme-containing protein. In some embodiments, a heterologous protein hasfunctional activity as a biocatalyst, as a food processing aid, anenzyme, as a flavor enhancer, a therapeutic, a sweetener, apharmaceutical, a nutraceutical.

As described herein, maintaining a pH between 8.5 and 12.0 during apurification process can result in a protein composition having minimaloff-flavors or off-odors such that the source of the protein (i.e., themicrobe from which the protein was purified) is not readilyidentifiable. In some embodiments, such a protein composition providesminimal off-flavors or off-odors to a food product of which it is a partor to which it is added. In some embodiments, off-flavor and off-odorgeneration can be assessed using trained human panelists. Theevaluations can involve eyeing, feeling, chewing, and/or tasting of theprotein or a food product made with the protein, to judge appearance,color, integrity, texture, flavor, and mouth feel, etc. Panelists can beserved samples under different colored lights (e.g., red or under whitelight). Samples can be assigned random three digit numbers and rotatedin ballot position to prevent bias. Panelists can be asked to correctlypair two different sample replicate sets (e.g., A1, A2 vs. B1, B2) in asample-blinded “tetrad” format. Panelists can be asked to correctly pairtwo different sample replicate sets (e.g., A1, A2, A3 vs. B1, B2, B3) ina sample-blinded “hexad” format. Sensory judgments can be scaled for“acceptance” or “likeability” or use special terminology. For example,letter scales (A for excellent, B for good, C for poor) or number scalescan be used (1=dislike, 2=fair, 3=good; 4=very good; 5=excellent). Ascale can be used to rate the overall acceptability or quality of thetested product or specific quality attributes such as beefiness,texture, and flavor. Panelists can be trained using specific sensoryreferences (e.g., “toasted grain” against a commercially availablecereal, or “fermented dairy” against a commercially available yogurt).Panelists can be given opportunity to comment on each sample andencouraged to rinse their mouths with water between samples.

In some embodiments, a protein composition described herein or a foodproduct made with such proteins can be assessed based upon olfactometerreadings. In various embodiments, the olfactometer can be used to assessodor concentration and odor thresholds, odor suprathresholds withcomparison to a reference gas, hedonic scale scores to determine thedegree of appreciation, or relative intensity of odors. In someembodiments, an olfactometer allows the training and automaticevaluation of expert panels.

In some embodiments, a protein composition can be used as a biocatalyst.For example, the substrate of an enzyme present in a protein compositioncan be added to the composition and, after incubation, the product ofthe enzymatic reaction can be isolated. In some embodiments, multiplesubstrates and cofactors may be added to support the production of oneor more products of interest. In some embodiments, the products of thereaction can a pharmaceutical, a pharmaceutical intermediate, a flavorcompound, a cofactor, a modified sugar, an amino acid, a monomer or anyother compound of interest.

In some embodiments, a protein composition can be used for in vitrotranscription and translation of proteins. In some embodiments, aprotein composition can be used for in vitro translation of proteins.Addition of template DNA, energy system and amino acids can lead toproduction of the target protein (see for example recent reviews such asMini-review: In vitro Metabolic Engineering for Biomanufacturing ofHigh-value Products Computational and Structural Biotechnology Journal,2017, Volume 15, 161-167). In some embodiments, a protein compositioncan be used to incorporate unnatural amino acids into proteins using invitro translation.

In some embodiments, a protein composition (e.g., produced by a methoddescribed herein) can comprise total cellular protein. In someembodiments, a protein composition (e.g., produced by a method describedherein) can comprise a plurality of proteins. For example, a proteincomposition can comprise 5 or more (e.g., 10, 15, 20, 30, 40, 50, 100,200, 300, 400, or 500) different proteins. In some embodiments, at least25% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the proteinin a protein composition is functional, as described herein.

Food Products

Any of the protein compositions described herein can be used as or inone or more food products. Protein compositions as described herein beused in a variety of food products, including, for example, proteinsupplements (e.g., protein powders or shakes), meal replacements, orbaked goods, or to replace all or a portion of an animal protein (e.g.,from cows, pigs, poultry, lamb, or fish) in a food product that mimicsan animal derived food product (e.g., a dairy replica (e.g., a milkreplica, a cheese replica), an egg replica (e.g., an albumen replica, anegg yolk replica, a whole egg replica, or a scrambled egg replica) ormeat replica such as a beef replica, a chicken replica, a pork replica,a fish replica, a lamb replica, any of which can be in the form of aground meat replica, a whole cut replica (e.g., a roast replica, steakreplica, a breast replica, wing replica, a thigh replica, a filetreplica, or a chop replica), an organ replica, or a sausage replica. Insome embodiments, a protein composition can be used as a meat extender.

In some embodiments, a protein composition as described herein can haveminimal off-flavors or off-odors such that the source of the protein(i.e., the microbe from which the protein was purified) is not readilyidentifiable and provide minimal off-flavors or off-odors to the foodproduct. In some embodiments, off-flavor and off-odor generation can beassessed using trained human panelists. The evaluations can involveeyeing, feeling, chewing, and/or tasting of the protein or a foodproduct made with the protein, to judge appearance, color, integrity,texture, flavor, and mouth feel, etc. Panelists can be served samplesunder different colored lights (e.g., red or under white light). Samplescan be assigned random three digit numbers and rotated in ballotposition to prevent bias. Sensory judgments can be scaled for“acceptance” or “likeability” or use special terminology. For example,letter scales (A for excellent, B for good, C for poor) or number scalescan be used (1=dislike, 2=fair, 3=good; 4=very good; 5=excellent). Ascale can be used to rate the overall acceptability or quality of thetested product or specific quality attributes such as beefiness,texture, and flavor. Panelists can be given opportunity to comment oneach sample and encouraged to rinse their mouths with water betweensamples.

In some embodiments, a protein composition described herein or a foodproduct made with such proteins can be assessed based upon olfactometerreadings. In various embodiments, the olfactometer can be used to assessodor concentration and odor thresholds, odor suprathresholds withcomparison to a reference gas, hedonic scale scores to determine thedegree of appreciation, or relative intensity of odors. In someembodiments, an olfactometer allows the training and automaticevaluation of expert panels.

In some embodiments, a protein composition described herein can compriseleast about 35% (e.g. at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 99%) by dry weight molecules larger than about500 Da (e.g., about 1 kDa, 2 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, or 50kDa). In some embodiments, a protein composition described herein cancomprise least about 35% (e.g. at least 40%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 99%) by dry weight molecules between about500 Da (e.g., about 1 kDa, 2 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, or 50kDa) and about 200 kDa (e.g., 300 kDa, 400 kDa, or 500 kDa). In someembodiments, at least about 35% (e.g. at least 40%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 99%) of the polypeptides (also calledproteins) in a protein composition described herein can fall betweenabout 500 Da (e.g., about 1 kDa, 2 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa, or50 kDa) and about 200 kDa (e.g., 300 kDa, 400 kDa, or 500 kDa). In someembodiments, a protein composition described herein can exclude one ormore small molecules that contribute to off-odors or off-flavors (e.g.,a protein composition can comprise no cysteine, 1-Hexanol; 2-Butylfuran;2-methyl-2-Pentenal; 3-Octanone; Ethyl-Acetate; 2-Ethyl-Furan;2-pentyl-Furan; Pyrazine; 1-Decanol; Acetophenone; 1-Nonanol;2,5-Dimethyl-Pyrazine; Dodecanal; Benzeneacetaldehyde; Nonanal;Butyrolactone; Octanal; 2-Decanone; Hexanal; 2-Nonanone; Benzaldehyde;Heptanal; 2-Octanone; Furfural; 2-Heptanone; Pentanal). A person ofordinary skill in the art can determine the total amount of smallmolecules or the amount of a particular small molecule in a sample,using, e.g., GCMS.

In some embodiments, a protein composition described herein can comprisea molecule that contribute to off-odors or off-flavors (e.g., cysteine,1-Hexanol; 2-Butylfuran; 2-methyl-2-Pentenal; 3-Octanone; Ethyl-Acetate;2-Ethyl-Furan; 2-pentyl-Furan; Pyrazine; 1-Decanol; Acetophenone;1-Nonanol; 2,5-Dimethyl-Pyrazine; Dodecanal; Benzeneacetaldehyde;Nonanal; Butyrolactone; Octanal; 2-Decanone; Hexanal; 2-Nonanone;Benzaldehyde; Heptanal; 2-Octanone; Furfural; 2-Heptanone; Pentanal). Aperson of ordinary skill in the art can determine the amount of aparticular small molecule in a sample, using, e.g., GCMS.

In some embodiments, a protein composition described herein can have abuffering capacity of less than about 2.5 mmol NaOH per gram dry solids(e.g., less than about 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8. 1.7, 1.6, 1.5,1.4, 1.3, 1.2, 1.1, 1.0, 0.5, or 0.1 mmol NaOH per gram dry solids). Thebuffer capacity can be determined by pH titration of a 2% (w/v)suspension or solution, measuring the mmol of NaOH that would berequired to move the suspension or solution from pH 3.0 to pH 12.0.

In some embodiments, a protein composition described herein can be inthe form of a solution. In some embodiments, a protein composition canbe in the form of a solid (e.g., a solution that has been freeze-driedor spray-dried). In some embodiments, a protein composition describedherein can be pasteurized. For example, a protein composition can bepasteurized by heat treatment, high temperature short timepasteurization, pulsed electric field, high pressure pasteurization, UVirradiation, gamma irradiation, or microfiltration. In some embodiments,one or more antimicrobials (e.g., polylysine) can be included in aprotein composition described herein.

In some embodiments, a protein composition described herein can be usedas a biocatalyst. For example, the substrate of an enzyme present in aprotein composition described herein can be added to the composition andafter incubation the product of the enzymatic reaction can be isolated.In some embodiments, multiple substrates and cofactors may be added tosupport the production of one or more products of interest. In someembodiments, the products of the reaction can a pharmaceutical, apharmaceutical intermediate, a flavor compound, a cofactor, a modifiedsugar, an amino acid, a monomer or any other compound of interest.

In some embodiments, a protein composition can be used for in vitrotranscription and translation of proteins. In some embodiments, aprotein composition can be used for in vitro translation of proteins.Addition of the template DNA, energy system and amino acids leads toproduction of the target protein (see for example recent reviews such asMini-review: In vitro Metabolic Engineering for Biomanufacturing ofHigh-value Products Computational and Structural Biotechnology Journal,2017, Volume 15, 161-167). In some embodiments, a protein compositioncan be used to incorporate unnatural amino acids into proteins using invitro translation.

Food products containing any of protein compositions described hereincan be used as a base for formulating a variety of additional foodproducts, including meat replicas, soup bases, stew bases, snack foods,bouillon powders, bouillon cubes, flavor packets, or frozen foodproducts. Meat replicas can be formulated, for example, as hot dogs,burgers, ground meat, sausages, steaks, filets, organs (such as liver,heart, tongue, kidney, sweetmeats, etc.) roasts, breasts, thighs, wings,meatballs, meatloaf, bacon, strips, fingers, nuggets, cutlets, or cubes.

Exemplary food products are described in U.S. Pat. Nos. 10,039,306,9,700,067, and 9,011,949; U.S. Patent Application Publication Nos.US20150305361A1, US20170172169A1, US20150289541A1, and US20170188612A1,each of which is incorporated by reference in its entirety.

In some embodiments, a food product can be a protein supplement. Forexample, in some embodiments, a protein composition as disclosed hereincan be part of a protein powder, which can be used in protein shakes,smoothies, baking, and the like.

In some embodiments, a food product can include a muscle replica. Insome embodiments, a food product can include an adipose replica. In someembodiments, a food product can include a muscle replica and an adiposereplica. In some embodiments, a food product that includes a musclereplica and an adipose replica can also be called a meat replica.

In some embodiments, a food product can be a dairy replica. In someembodiments, a food product can be a cheese replica. In someembodiments, a food product can be a milk replica. In some embodiments,a milk replica can be used to make a cheese replica.

In some embodiments, a food product can be an egg replica. In someembodiments, a food product can be a whole egg replica (e.g., with ayolk replica partitioned from an albumen replica). In some embodiments,a food product can be an egg yolk replica. In some embodiments, a foodproduct can be an albumen replica. In some embodiments, a food productcan be a scrambled egg replica (e.g., a mixture of an egg yolk replicaand an albumen replica).

A food product can include one or more proteins (e.g., a proteincomposition as described herein, a commercially available protein, aprotein purified by any method known in the art, or a combinationthereof). In some embodiments, a food product can include any of theprotein compositions as described herein. In some embodiments, a foodproduct can include any of the protein compositions as described hereinin addition to a commercially available protein (e.g., soy proteinconcentrate, soy protein isolate, casein, whey, wheat gluten, peavicilin, or pea legumin). In some embodiments, a food product caninclude any of the protein compositions as described herein, in additionto one or more proteins purified by any method known in the art.

One or more proteins (e.g., a protein composition as described herein, acommercially available protein, a protein purified by any method knownin the art, or a combination thereof) can be present in an amount ofabout 0.1% to about 100% by weight (e.g., about 0.1% to about 1%, about1% to about 5%, about 5% to about 10%, about 1% to about 10%, about 10%to about 20%, about 20% to about 30%, about 30% to about 40%, about 40%to about 50%, about 50% to about 60%, about 60% to about 70%, about 70%to about 80%, about 80% to about 90%, about 90% to about 100% about 10%to about 30%, about 30% to about 50%, about 50% to about 70%, about 70%to about 90%, about 0.1% to about 20%, about 20% to about 40%, about 40%to about 60%, about 60% to about 80%, about 80% to about 100%, about0.1% to about 33%, about 33% to about 66%, about 66% to about 100, about0.1% to about 50%, or about 50% to about 100%) of a food product (e.g.,a meat replica, a beef-like food product, a chicken-like food product, apork-like food product, a fish-like food product, a beef food product, achicken food product, a pork food product, or a fish food product).

Any of the food products described herein can include an iron complex(e.g., ferrous chlorophyllin (e.g., CAS No. 69138-22-3), ironpheophorbide (e.g., CAS No. 15664-29-6), an iron salt (e.g. iron sulfate(e.g., any of CAS Nos. 7720-78-7, 17375-41-6, 7782-63-0, or 10028-22-5)iron gluconate (e.g., any of CAS Nos. 299-29-6, 22830-45-1, or699014-53-4), iron citrate (e.g., any of CAS Nos. 3522-50-7, 2338-05-8,or 207399-12-0), ferric EDTA (e.g., CAS No. 17099-81-9) or a heme moietysuch as a heme (e.g., heme A (e.g., CAS No. 18535-39-2), heme B (e.g.CAS No. 14875-96-8), heme C (e.g., CAS No. 26598-29-8), heme 0 (e.g.,CAS No. 137397-56-9), heme I, heme M, heme D, heme S) or aheme-containing protein). For example, a structure of heme B is shown inFIG. 8 .

In some embodiments, the heme moiety is a heme noncovalently orcovalently bound to a protein or polypeptide as a heme-containingprotein. In some embodiments, the protein or polypeptide is a globin. Insome embodiments, the globin is selected from the group consisting of anandroglobin, a cytoglobin, a globin E, a globin X, a globin Y, ahemoglobin, a myoglobin, a leghemoglobin, an erythrocruorin, a betahemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, acytoglobin, a histoglobin, a neuroglobins, a chlorocruorin, a truncatedhemoglobin, a truncated 2/2 globin, and a hemoglobin 3. In someembodiments, the protein or polypeptide is a non-animal protein orpolypeptide. In some embodiments, the protein or polypeptide is a plant,fungal, algal, archaeal, or bacterial protein. In some embodiments, theprotein or polypeptide is not natively expressed in plant, fungal,algal, archaeal, or bacterial cells. In some embodiments, the protein orpolypeptide comprises an amino acid sequence having at least 50%sequence identity (e.g., at least 60%, 70%, 80%, 90%, or 95% sequenceidentity) to a polypeptide set forth in SEQ ID NOs. 1-27.

Heme-containing proteins that can be used in any of the food productsdescribed herein can be from mammals (e.g., farms animals such as cows,goats, sheep, pigs, ox, or rabbits), birds, plants, algae, fungi (e.g.,yeast or filamentous fungi), ciliates, or bacteria. For example, aheme-containing protein can be from a mammal such as a farm animal(e.g., a cow, goat, sheep, pig, ox, or rabbit) or a bird such as aturkey or chicken. Heme-containing proteins can be from a plant such asNicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (corn),Arabidopsis thaliana, a legume such as Glycine max (soybean), Cicerarietinum (garbanzo or chick pea), Pisum sativum (pea) varieties such asgarden peas or sugar snap peas, Phaseolus vulgaris varieties of commonbeans such as green beans, black beans, navy beans, northern beans, orpinto beans, Vigna unguiculata varieties (cow peas), Vigna radiata (Mungbeans), Lupinus albus (lupin), or Medicago sativa (alfalfa); Brassicanapus (canola); Triticum sps. (wheat, including wheat berries, andspelt); Gossypium hirsutum (cotton); Oryza sativa (rice); Zizania sps.(wild rice); Helianthus annuus (sunflower); Beta vulgaris (sugarbeet);Pennisetum glaucum (pearl millet); Chenopodium sp. (quina); Sesamum sp.(sesame); Linum usitatissimum (flax); or Hordeum vulgare (barley).Heme-containing proteins can be isolated from fungi such asSaccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusariumgraminearum, Aspergillus oryzae, Trichoderma reesei, Mycelioptherathermophile, Kluyvera lactis, or Fusarium oxysporum. Heme-containingproteins can be isolated from bacteria such as Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium,Synechocistis sp., Aquifex aeolicus, Methylacidiphilum infernorum, orthermophilic bacteria such as Thermophilus. The sequences and structureof numerous heme-containing proteins are known. See for example, Reedy,et al., Nucleic Acids Research, 2008, Vol. 36, Database issue D307-D313and the Heme Protein Database available on the world wide web athemeprotein.info/heme.php.

For example, a non-symbiotic hemoglobin can be from a plant selectedfrom the group consisting of soybean, sprouted soybean, alfalfa, goldenflax, black bean, black eyed pea, northern, garbanzo, moong bean,cowpeas, pinto beans, pod peas, quinoa, sesame, sunflower, wheatberries, spelt, barley, wild rice, or rice.

Any of the heme-containing proteins described herein that can be usedfor producing food products can have at least 70% (e.g., at least 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to theamino acid sequence of the corresponding wild-type heme-containingprotein or fragments thereof that contain a heme-binding motif. Forexample, a heme-containing protein can have at least 70% sequenceidentity to an amino acid sequence, including a non-symbiotic hemoglobinsuch as that from Vigna radiata (SEQ ID NO:1), Hordeum vulgare (SEQ IDNO:5), Zea mays (SEQ ID NO:13), Oryza sativa subsp. japonica (rice) (SEQID NO:14), or Arabidopsis thaliana (SEQ ID NO:15), a Hell's gate globinI such as that from Methylacidiphilum infernorum (SEQ ID NO:2), aflavohemoprotein such as that from Aquifex aeolicus (SEQ ID NO:3), aleghemoglobin such as that from Glycine max (SEQ ID NO:4), Pisum sativum(SEQ ID NO:16), or Vigna unguiculata (SEQ ID NO:17), a heme-dependentperoxidase such as from Magnaporthe oryzae, (SEQ ID NO:6) or Fusariumoxysporum (SEQ ID NO:7), a cytochrome c peroxidase from Fusariumgraminearum (SEQ ID NO:8), a truncated hemoglobin from Chlamydomonasmoewusii (SEQ ID NO:9), Tetrahymena pyriformis (SEQ ID NO:10, group Itruncated), Paramecium caudatum (SEQ ID NO:11, group I truncated), ahemoglobin from Aspergillus niger (SEQ ID NO:12), or a mammalianmyoglobin protein such as the Bos taurus (SEQ ID NO:18) myoglobin, Susscrofa (SEQ ID NO:19) myoglobin, Equus caballus (SEQ ID NO:20)myoglobin, a heme-protein from Nicotiana benthamiana (SEQ ID NO:21),Bacillus subtilis (SEQ ID NO:22), Corynebacterium glutamicum (SEQ IDNO:23), Synechocystis PCC6803 (SEQ ID NO:24), Synechococcus sp. PCC 7335(SEQ ID NO:25), Nostoc commune (SEQ ID NO:26), or Bacillus megaterium(SEQ ID NO: 27).

The percent identity between two amino acid sequences can be determinedas follows. First, the amino acid sequences are aligned using the BLAST2 Sequences (B12seq) program from the stand-alone version of BLASTZcontaining BLASTP version 2.0.14. This stand-alone version of BLASTZ canbe obtained from Fish & Richardson's web site (e.g., fr.com/blast/) orthe U.S. government's National Center for Biotechnology Information website (ncbi.nlm.nih.gov). Instructions explaining how to use the B12seqprogram can be found in the readme file accompanying BLASTZ. B12seqperforms a comparison between two amino acid sequences using the BLASTPalgorithm. To compare two amino acid sequences, the options of B12seqare set as follows: -i is set to a file containing the first amino acidsequence to be compared (e.g., C:\seq1.txt); -j is set to a filecontaining the second amino acid sequence to be compared (e.g.,C:\seq2.txt); -p is set to blastp; -o is set to any desired file name(e.g., C:\output.txt); and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison between two amino acid sequences:C:\B12seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. Ifthe two compared sequences share homology, then the designated outputfile will present those regions of homology as aligned sequences. If thetwo compared sequences do not share homology, then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity is determined by dividing the number ofmatches by the length of the full-length polypeptide amino acid sequencefollowed by multiplying the resulting value by 100. It is noted that thepercent identity value is rounded to the nearest tenth. For example,78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15,78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is notedthat the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

In some embodiments, heme-containing proteins can be extracted from aproduction organism (e.g., extracted from animal tissue, or plant,fungal, algal, or bacterial biomass, or from the culture supernatant forsecreted proteins) or from a combination of production organisms (e.g.,multiple plant species). Leghemoglobin is readily available as an unusedby-product of commodity legume crops (e.g., soybean, alfalfa, or pea).The amount of leghemoglobin in the roots of these crops in the UnitedStates exceeds the myoglobin content of all the red meat consumed in theUnited States.

In some embodiments, extracts of heme-containing proteins include one ormore non-heme-containing proteins from the source material (e.g., otheranimal, plant, fungal, algal, or bacterial proteins) or from acombination of source materials (e.g., different animal, plant, fungi,algae, or bacteria). For example, a heme-containing protein can be partof a protein composition as described herein.

In some embodiments, heme-containing proteins can be provided in a foodproduct in a form that is not part of a protein composition as describedherein. In some embodiments, heme-containing proteins can be purified byany method known in the art.

Proteins can be separated on the basis of their molecular weight, forexample, by size exclusion chromatography, ultrafiltration throughmembranes, or density centrifugation. In some embodiments, the proteinscan be separated based on their surface charge, for example, byisoelectric precipitation, anion exchange chromatography, or cationexchange chromatography. Proteins also can be separated on the basis oftheir solubility, for example, by ammonium sulfate precipitation,isoelectric precipitation, surfactants, detergents or solventextraction. Proteins also can be separated by their affinity to anothermolecule, using, for example, hydrophobic interaction chromatography,reactive dyes, or hydroxyapatite. Affinity chromatography also caninclude using antibodies having specific binding affinity for theprotein (e.g., the heme-containing protein), nickel NTA for His-taggedrecombinant proteins, lectins to bind to sugar moieties on aglycoprotein, or other molecules which specifically binds the protein.

Heme-containing proteins also can be recombinantly produced usingpolypeptide expression techniques (e.g., heterologous expressiontechniques using bacterial cells, insect cells, fungal cells such asyeast, plant cells such as tobacco, soybean, or Arabidopsis, ormammalian cells). In some cases, standard polypeptide synthesistechniques (e.g., liquid-phase polypeptide synthesis techniques orsolid-phase polypeptide synthesis techniques) can be used to produceheme-containing proteins synthetically. In some cases, in vitrotranscription-translation techniques can be used to produceheme-containing proteins.

In some embodiments, a heme-containing protein is part of a totalcellular protein composition as described herein.

A heme-containing protein can be present in a food product (e.g., adairy replica, a cheese replica, an egg replica, a meat replica, abeef-like food product, a chicken-like food product, a pork-like foodproduct, a fish-like food product, a beef food product, a chicken foodproduct, a pork food product, or a fish food product) in an amount ofabout 0.005% to about 5% (wt heme-containing protein/wt food product)(e.g., about 0.005% to about 0.01%, about 0.01% to about 0.1%, about0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 1% toabout 3%, about 3% to about 5%, or about 1% to about 5% (wt/wt)). Insome embodiments, a heme-containing protein can be a non-animalheme-containing protein. In some embodiments, a heme-containing proteincan be an algal, bacterial, fungal, plant, or Archaeal heme-containingprotein.

A heme can be present in a food product (e.g., a dairy replica, a cheesereplica, an egg replica, a meat replica, a beef-like food product, achicken-like food product, a pork-like food product, a fish-like foodproduct, a beef food product, a chicken food product, a pork foodproduct, or a fish food product) in an amount of about 0.00005% to about2% (wt heme/wt food product) (e.g., about 0.00005% to about 0.0001%,about 0.0001% to about 0.0005%, about 0.0005% to about 0.001%, about0.001% to about 0.005%, about 0.005% to about 0.01%, about 0.01% toabout 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about0.5% to about 1%, about 0.1% to about 0.2%, about 0.2% to about 0.4%,about 0.4% to about 0.6%, about 0.6% to about 0.8%, about 0.8% to about1%, about 1% to about 2%, about 1.0% to about 1.2%, about 1.2% to about1.4%, about 1.4% to about 1.6%, about 1.6% to about 1.8%, or about 1.8%to about 2.0% (wt/wt)).

Food products described herein can be free of or substantially free ofsome types of animal products (e.g., animal heme-containing proteins, orall animal products).

In some embodiments, a food product can be substantially soy-free,substantially wheat-free, substantially yeast-free, substantiallyMSG-free, substantially free of protein hydrolysis products, soy-free,wheat-free, yeast-free, MSG-free, and/or free of protein hydrolysisproducts, and can taste meaty, highly savory, and without off odors orflavors.

In some embodiments, a food product can include one or more flavorprecursors. Suitable flavor precursors include sugars, sugar alcohols,sugar derivatives, oils (e.g., vegetable oils), free fatty acids,alpha-hydroxy acids, dicarboxylic acids, amino acids and derivativesthereof, nucleosides, nucleotides, vitamins, peptides, proteinhydrolysates, extracts, phospholipids, lecithin, and organic molecules.Non-limiting examples of such flavor precursors are provided in Table 1.

TABLE 1 Flavor Precursor Molecules Sugars, sugar alcohols, sugar acids,and sugar derivatives: glucose, fructose, ribose, sucrose, arabinose,glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-diphosphate,inositol, maltose, molasses, maltodextrin, glycogen, galactose, lactose,ribitol, gluconic acid and glucuronic acid, amylose, amylopectin, orxylose Oils: coconut oil, mango oil, sunflower oil, cottonseed oil,safflower oil, rice bran oil, cocoa butter, palm fruit oil, palm oil,soybean oil, canola oil, corn oil, sesame oil, walnut oil, flaxseed,jojoba oil, castor, grapeseed oil, peanut oil, olive oil, algal oil, oilfrom bacteria or fungi Free fatty acids: caprylic acid, capric acid,lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic,oleic acid, linoleic acid, alpha linolenic acid, gamma linolenic acid,arachidic acid, arachidonic acid, behenic acid, or erucic acid Aminoacids and derivatives thereof: cysteine, cystine, a cysteine sulfoxide,allicin, selenocysteine, methionine, isoleucine, leucine, lysine,phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine,arginine, histidine, alanine, asparagine, aspartate, glutamate,glutamine, glycine, proline, serine, or tyrosine Nucleosides andNucleotides: inosine, inosine monophosphate (IMP), guanosine, guanosinemonophosphate (GMP), adenosine, adenosine monophosphate (AMP) Vitamins:thiamine, vitamin C, Vitamin D, Vitamin B6, or Vitamin E Misc:phospholipid, lecithin, pyrazine, creatine, pyrophosphate Acids: aceticacid, alpha hydroxy acids such as lactic acid or glycolic acid,tricarboxylic acids such as citric acid, dicarboxylic acids such assuccinic acid or tartaric acid Peptides and protein hydrolysates:glutathione, vegetable protein hydrolysates, soy protein hydrolysates,yeast protein hydrolysates, algal protein hydrolysates, meat proteinhydrolysates Extracts: a malt extract, a yeast extract, and a peptone

Food products described herein can be packaged in various ways,including being sealed within individual packets or shakers, such thatthe composition can be sprinkled or spread on top of a food productbefore or during cooking.

Food products described herein can include additional ingredientsincluding food-grade oils such as canola, corn, sunflower, soybean,olive or coconut oil, seasoning agents such as edible salts (e.g.,sodium or potassium chloride) or herbs (e.g., rosemary, thyme, basil,sage, or mint), flavoring agents, proteins (e.g., soy protein isolate,wheat glutin, pea vicilin, and/or pea legumin), protein concentrates(e.g., soy protein concentrate), emulsifiers (e.g., lecithin), gellingagents (e.g., k-carrageenan or gelatin), fibers (e.g., bamboo filer orinulin), or minerals (e.g., iodine, zinc, and/or calcium).

Food products described herein also can include a natural coloring agentsuch as turmeric or beet juice, or an artificial coloring agent such asazo dyes, triphenylmethanes, xanthenes, quinines, indigoids, titaniumdioxide, red #3, red #40, blue #1, or yellow #5.

Food products described herein also can include meat shelf lifeextenders such as carbon monoxide, nitrites, sodium metabisulfite,Bombal, vitamin E, rosemary extract, green tea extract, catechins andother anti-oxidants.

In some embodiments, a food product including a heme, a flavorprecursor, or a combination thereof, when cooked, can result in theincreased production of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,or more) volatile compounds associated with a meat-like aroma.Non-limiting examples of volatile compounds associated with a meat-likearoma are presented in the attached Appendix 1, each of which has beenassociated with meat aroma, such as the aroma of beef, chicken, or pork,as supported by the listed references. In some embodiments, cooking anyof the food products as described herein can result in increasedproduction of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,18, 20 or more) volatile compounds selected from the group consisting of(E)-2-decenal, (E)-2-heptenal, (E)-2-nonenal, (E)-2-octen-1-ol,(E)-2-octenal, (E)-3-penten-2-one, (E,E)-2,4-hexadienal,1-(2-furanyl)-ethanone, 1-(acetyloxy)-2-propanone, 1-heptanol,1-hexanol, 1-octanol, 1-penten-3-one, 1-undecanol,2,3-dimethyl-pyrazine, 2,3-hexanedione, 2,4-dimethyl-thiazole,2,5-dimethyl-pyrazine, 2,6-dimethylpyrazine, 2-acetyl-1-pyrroline,2-acetylthiazole, 2-butanol, 2-butanone, 2-butenal, 2-heptanone,2-hydroxy-benzaldehyde, 2-methyl-2(E)-butenal, 2-methyl-3-furanthiol,2-methyl-butanal, 2-methyl-propanal, 2-methyl-thiazole, 2-n-butyl furan,2-pentyl-furan, 2-propenal, 2-undecanone, 3-ethyl-pyridine,3-methyl-2-butenal, 3-methyl-3-buten-2-one, 3-methyl-butanal,3-methyl-hexane, 3-methyl-thiophene, 4-pentenal,5-methyl-2-thiophenecarboxaldehyde, 6-methyl-5-hepten-2-one,acetaldehyde, acetone, acetophenone, benzaldehyde, benzeneacetaldehyde,bis(2-methyl-3-furyl)disulfide, dimethyl disulfide, dimethyl trisulfide,dodecanal, E-2-undecenal, ethyl-pyrazine, furan, furfural, heptanal,hexanal, methional, methyl-thiirane, propyl mercaptan, pyrazine,pyridine, tetradecane, tetrahydro-2H-pyran-2-one, andtrimethyl-pyrazine.

Food products described herein can include a lipid (also called a fat)component. Lipids can be isolated and/or purified and can be in the formof triglycerides, monoglycerides, diglycerides, free fatty acids,sphingosides, glycolipids, phospholipids, or oils, or assemblies of suchlipids (e.g., membranes, lecithin, lysolecithin, or fat dropletscontaining a small amount of lipid in a bulk water phase). In someembodiments, lipid sources are oils obtained from non-animal sources(e.g., oils obtained from plants, algae, fungi such as yeast orfilamentous fungi, seaweed, bacteria, or Archae), including geneticallyengineered bacteria, algae, archaea or fungi. Non-limiting examples ofplant oils include corn oil, olive oil, soy oil, peanut oil, walnut oil,almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil,safflower oil, sunflower oil, flax seed oil, palm oil, palm kernel oil,coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheatgerm oil, or rice bran oil; or margarine. Oils can be hydrogenated(e.g., a hydrogenated vegetable oil) or non-hydrogenated.

In some embodiments, a lipid can be triglycerides, monoglycerides,diglycerides, free fatty acids, sphingosides, glycolipids, lecithin,lysolecithin, phospholipids such as phosphatidic acids, lysophosphatidicacids, phosphatidyl cholines, phosphatidyl inositols, phosphatidylethanolamines, or phosphatidyl serines; sphingolipids such assphingomyelins or ceramides; sterols such as stigmasterol, sitosterol,campesterol, brassicasterol, sitostanol, campestanol, ergosterol,zymosterol, fecosterol, dinosterol, lanosterol, cholesterol, orepisterol; lipid amides, such as N-palmitoyl proline, N-stearoylglycine, N-palmitoyl glycine, N-arachidonoyl glycine, N-palmitoyltaurine, N-arachidonoyl histidine, or anandamide; free fatty acids suchas palmitoleic acid, palmitic acid, myristic acid, lauric acid,myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonicacid, undecanoic acid, linoleic acid (C18:2), eicosanoic acid (C22:0),arachidonic acid (C20:4), eicosapentanoic acid (C20:5), docosapentaenoicacid (C22:5), docosahexanoic acid (C22:6), erucic acid (C22:1),conjugated linoleic acid, linolenic acid (C18:3), oleic acid (C18:1),elaidic acid (trans isomer of oleic acid), trans-vaccenic acid (C18:1trans 11), or conjugated oleic acid; or esters of such fatty acids,including monoacylglyceride esters, diacylglyceride esters, andtriacylglyceride esters of such fatty acids.

Lipids can comprise phospholipids, lipid amides, sterols, or neutrallipids. The phospholipids can comprise a plurality of amphipathicmolecules comprising fatty acids, glycerol and polar groups. In someembodiments, the polar groups are, for example, choline, ethanolamine,serine, phosphate, glycerol-3-phosphate, inositol or inositolphosphates. In some embodiments, lipids are, for example, sphingolipids,ceramides, sphingomyelins, cerebrosides, gangliosides, ether lipids,plasmalogens or pegylated lipids.

In some embodiments, a fat can be present in an amount of about 0.1% toabout 95% by weight (e.g., about 0.1% to about 1%, about 1% to about 5%,about 5% to about 10%, about 1% to about 10%, about 10% to about 20%,about 20% to about 30%, about 30% to about 40%, about 40% to about 50%,about 50% to about 60%, about 60% to about 70%, about 70% to about 80%,about 80% to about 90%, about 90% to about 95% about 10% to about 30%,about 30% to about 50%, about 50% to about 70%, about 70% to about 90%,about 0.1% to about 20%, about 20% to about 40%, about 40% to about 60%,about 60% to about 80%, about 80% to about 95%, about 0.1% to about 33%,about 33% to about 66%, about 66% to about 95%, about 0.1% to about 50%,or about 50% to about 95%) of a food product (e.g., a meat replica, abeef-like food product, a chicken-like food product, a pork-like foodproduct, a fish-like food product, a beef food product, a chicken foodproduct, a pork food product, or a fish food product).

In some embodiments, a fat can be present in a food product in the formof an adipose replica.

A food product can include a binding agent or a carbohydrate-based gel.In some embodiments, a carbohydrate-based gel can be included in abinding agent. A binding agent can be about 2% to about 10% by weight ofa food product. A binding agent can include one or more proteins thathave been chemically or enzymatically modified to improve their texturaland/or flavor properties, or to modify their denaturation and gellingtemperatures. A carbohydrate based gel can contain methylcellulose,hydroxypropylmethyl cellulose, guar gum, locust bean gum, xanthan gum,agar, pectin, carrageenan, konjac, alginate, chemically-modifiedagarose, or a mixture thereof. A binding agent can include egg albuminor collagen.

The disclosure provides, in certain embodiments, methods for determiningthe suitability for a consumable to qualify as a replica of a foodproduct, for example, by determining whether an animal or human candistinguish the consumable from a predicate food product, e.g., aparticular meat. One method to determine whether the consumable iscomparable to a food product (e.g. meat) is to a) define the propertiesof meat and b) determine whether the consumable has similar properties.

Properties that can be tested or used to compare or describe a foodproduct include mechanical properties such as hardness, cohesiveness,brittleness, chewiness, gumminess, viscosity, elasticity, andadhesiveness. Properties of food products that can be tested alsoinclude geometric properties such as particle size and shape, andparticle shape and orientation. The three dimensional organization ofparticles may also be tested. Additional properties can include moisturecontent and fat content. These properties can be described using termssuch as “soft,” “firm” or “hard” describe hardness; “crumbly,”“crunchy,” “brittle,” “chewy,” “tender,” “tough,” “short,” “mealy,”“pasty,” or “gummy,” to describe cohesiveness; “thin” or “viscous” todescribe viscosity; “plastic” or “elastic” to describe elasticity;“sticky,” “tacky” or “gooey” to describe adhesiveness; “gritty,”“grainy” or “course” to describe particle shape and size; “fibrous,”“cellular” or “crystalline” to describe particle shape and orientation,“dry,” “moist,” “wet,” or “watery” to describe moisture content; or“oily” or “greasy” to describe fat content. Accordingly, in someembodiments, a group of people can be asked to rate a certain referencefood product, for instance ground beef, according to properties whichdescribe the reference food product. A food product described herein canbe rated by the same people to determine equivalence.

Flavor of a food product of the disclosure can also be assessed. Flavorscan be rated according to similarity to reference foods, e.g., “eggy,”“fishy,” “buttery,” “chocolaty,” “fruity”, “peppery,” “baconlike,”“creamy,” “milky,” or “beefy.” Flavors can be rated according to theseven basic tastes, i.e., sweet, sour, bitter, salty, umami (savory),pungent (or piquant), and metallic. Flavors can be described accordingto the similarity to an experience caused by a chemical, e.g., diacetyl(buttery), 3-hydroxy-2 butanone (buttery), nona-2E-enal (fatty),1-octene-3-ol (mushroom), hexanoic acid (sweaty), 4-hydroxy-5-methylfuranone (HMF, meaty), pyrazines (nutty), bis(2-methyl-3-furyl)disulfide (roast meat), decanone (musty/fruity), isoamyl acetate(banana), benzaldehyde (bitter almond), cinnamic aldehyde (cinnamon),ethyl propionate (fruity), methyl anthranilate (grape), limonene(orange), ethyl decadienoate (pear), allyl hexanoate (pineapple), ethylmaltol (sugar, cotton candy), ethylvanillin (vanilla), butanoic acid(rancid), 12-methyltridecanal (beefy), or methyl salicylate(wintergreen). These ratings can be used as an indication of theproperties of the reference food product. A food product of the presentdisclosure can then be compared to a reference food product to determinehow similar the food product is to the reference food product. In someembodiments, the properties of a food product of the disclosure are thenaltered to make the food product of the disclosure more similar to thereference food product. Accordingly, in some embodiments, a food productof the disclosure is rated similar to a reference food product accordingto human evaluation. In some embodiments, a food product of thedisclosure is indistinguishable from the reference food product to ahuman.

In some embodiments, subjects asked to identify the food product of thedisclosure can identify it as a form of a reference food product, or asa particular reference food product, e.g., a subject will identify afood product of the disclosure as meat. For example, in someembodiments, a human can identify a food product of the disclosure ashaving properties equivalent to meat. In some embodiments, one or moreproperties of the food product of the disclosure are equivalent to thecorresponding properties of meat according to a human's perception. Suchproperties include the properties that can be tested. In someembodiments, a human identifies a food product of the present disclosureas more meat like than any meat replicas found in the art.

Experiments can demonstrate that a food product of the disclosure isacceptable to consumers. A panel can be used to screen a variety ofconsumables described herein. A number of human panelists can testmultiple food product samples, namely, natural meats vs. the foodproducts described herein, or a meat substitute vs. a consumablecomposition described herein. Variables such as fat content can bestandardized, for example to 20% fat using lean and fat meat mixes. Fatcontent can be determined using the Babcock for meat method (S. S.Nielson, Introduction to the Chemical Analysis of Foods (Jones &Bartlett Publishers, Boston, 1994)). Mixtures of ground beef and foodproducts of the invention prepared according to the procedure describedherein can be formulated.

Panelists can be served samples (e.g., in booths), under red lights orunder white light, in an open consumer panel. Samples can be assignedrandom three-digit numbers and rotated in ballot position to preventbias. Panelists can be asked to evaluate samples for tenderness,juiciness, texture, flavor, and overall acceptability using a hedonicscale from 1=dislike extremely, to 9=like extremely, with a median of5=neither like nor dislike. Panelists can be encouraged to rinse theirmouths with water between samples, and given opportunity to comment oneach sample.

The results of this experiment can indicate significant differences orsimilarities between the traditional meats and the food products of thedisclosure.

These results can demonstrate that the food products described hereinare judged as acceptably equivalent to real meat products. Additionally,these results can demonstrate that food products described herein arepreferred by panelists over other commercially available meatsubstitutes. Thus, in some embodiments, the present disclosure providesfor food products that are similar to traditional meats and are moremeat like than previously known meat alternatives.

Food products of the disclosure can also have similar physicalcharacteristics as food products, e.g., traditional meat. In oneembodiment, the force required to pierce a 1 inch thick structure (e.g.,a patty) made of a food product of the disclosure with a fixed diametersteel rod is not significantly different than the force required topierce a 1 inch thick similar food product structure (e.g., a groundbeef patty) with a similar fixed diameter steel rod. Accordingly, thedisclosure provides for food products with similar physical strengthcharacteristics to meat. In another embodiment, the force required totear a sample of a food product of the disclosure with a cross-sectionalarea of 100 mm² is not significantly different than the force requiredto tear a sample of animal tissue (muscle, fat or connective tissue)with a cross-sectional area 100 mm² measured the same way. Force can bemeasured using, for example, TA.XT Plus Texture Analyzer (TextrueTechnologies Corp.). Accordingly, the disclosure provides for foodproducts with similar physical strength characteristics to meat.

Food products described herein can have a similar cook losscharacteristic as a food product, e.g., meat. For example, a foodproduct of the disclosure can have a similar fat and protein content toground beef and have the same reduction in size when cooked as realground beef. Similarities in size loss profiles can be achieved forvarious compositions of food products described herein matched tovarious meats. The cook loss characteristics of a food product describedherein also can be engineered to be superior to food products. Forexample, a food product described herein can be produced that has lessloss during cooking but achieves similar tastes and texture qualities asthe cooked products. One way this can be achieved is by altering theproportions of lipids based on melting temperatures in a food product ofthe disclosure. Another way this can be achieved is by altering theprotein composition of a food product by controlling the concentrationof protein or by the mechanism by which a tissue replica is formed.

In some embodiments, a food product of the disclosure is compared to areference food product (e.g., an animal based food product (e.g., meat))based upon olfactometer readings. In some embodiments, an olfactometercan be used to assess odor concentration and odor thresholds, or odorsuprathresholds with comparison to a reference gas, hedonic scale scoresto determine the degree of appreciation, or relative intensity of odors.In some embodiments, an olfactometer allows the training and automaticevaluation of expert panels. So in some embodiments, a food product ofthe disclosure is a food product that causes similar or identicalolfactometer readings to a reference food product. In some embodiments,the differences are sufficiently small to be below the detectionthreshold of human perception.

Gas chromatography-mass spectrometry (GCMS) is a method that combinesthe features of gas-liquid chromatography and mass spectrometry toseparate and identify different substances within a test sample. GCMScan, in some embodiments, be used to evaluate the properties of a foodproduct of the disclosure. For example, volatile chemicals can beisolated from the head space around meat. These chemicals can beidentified using GCMS. A profile of the volatile chemicals in theheadspace around meat can be thereby created. In some embodiments, eachpeak of the GCMS can be further evaluated. For instance, a human couldrate the experience of smelling the chemical responsible for a certainpeak. This information could be used to further refine the profile. GCMScould then be used to evaluate the properties of the consumable. TheGCMS profile can be used to refine the consumable.

Characteristic flavor and fragrance components are mostly producedduring the cooking process by chemical reactions molecules includingamino acids, fats and sugars which are found in plants as well as meat.Therefore, in some embodiments, a food product of the disclosure istested for similarity to meat during or after cooking. In someembodiments human ratings, human evaluation, olfactometer readings, orGCMS measurements, or combinations thereof, are used to create anolfactory map of a reference food product (e.g., cooked meat).Similarly, an olfactory map of a food product of the disclosure, forinstance a meat replica, can be created. These maps can be compared toassess how similar the cooked consumable is to meat. In someembodiments, an olfactory map of a food product of the disclosure duringor after cooking is similar to or indistinguishable from that of cookedor cooking meat. In some embodiments, the similarity is sufficient to bebeyond the detection threshold of human perception. In some embodiments,a food product of the disclosure can be created so its characteristicsare similar to a reference food product after cooking, but the uncookedfood product of the disclosure can have properties that are differentfrom the reference food product prior to cooking.

In some embodiments, a food product can be a dairy replica (also calleda non-dairy product).

In one aspect, the disclosure provides a non-dairy cheese source thatcan be used as a starting material for preparing a non-dairy cheese. Theterm “non-dairy cheese source” refers to an emulsion comprising proteins(e.g., including a protein composition as described herein, acommercially available protein, or a protein purified by any methodknown in the art, or a combination thereof) and fats, wherein saidproteins and fats are prepared from a non-dairy source.

In some embodiments, a non-dairy cheese source can be a milk replica(also called a non-dairy milk). In some embodiments, a milk replica canbe used to make a cheese replica (also called a non-dairy cheese).

In some embodiments, a non-dairy milk is an emulsion comprising one ormore proteins (e.g., a protein composition as described herein, acommercially available protein, a protein purified by any method knownin the art, or a combination thereof) and one or more fats. In someembodiments, the proteins are contained in a protein solution. Thesolution can comprise EDTA (0-0.1M), NaCl (0-1M), KCl (0-1M), NaSO₄(0-0.2M), potassium phosphate (0-1M), sodium citrate (0-1M), sodiumcarbonate (0-1M), sucrose (0-50%), Urea (0-2M) or any combinationthereof. The solution can have a pH of 3 to 11. In some embodiments, theone or more proteins accounts for 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the protein content ofsaid protein solution. In some embodiments, the one or more proteinsaccounts for 0.1-5%, 1-10%, 5-20%, 10-40%, 30-60%, 40-80%, 50-90%,60-95%, or 70-100% of the protein content of said protein solution. Insome embodiments, the total protein content of the protein solution isabout 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.75%, 1%, 1.5%, 2%, 5%, 7.5%, 10%,12.5%, 15%, 17.5%, 20%, or more than 20% weight/volume. In someembodiments, the total protein content of the protein solution is0.1-5%, 1-10%, 5-20%, or more than 20% weight/volume.

In some embodiments, the proteins are concentrated using any methodsknown in the art. The proteins may be concentrated 2-fold, five-fold,10-fold, or up to 100 fold. The proteins may be concentrated to a finalconcentration of 0.001-1%, 0.05-2%, 0.1-5%, 1-10%, 2-15%, 4-20%, or morethan 20%. Exemplary methods include, e.g., ultrafiltration (ortangential flow filtration), lyophilisation, spray drying, or thin filmevaporation.

In some embodiments, fats used in preparing the emulsion can be from avariety of sources. In some embodiments, the sources can be non-animalsources (e.g., oils obtained from plants, algae, fungi such as yeast orfilamentous fungi, seaweed, bacteria, Archae), including geneticallyengineered bacteria, algae, archaea or fungi. The oils can behydrogenated (e.g., a hydrogenated vegetable oil) or non-hydrogenated.Non-limiting examples of plant oils include corn oil, olive oil, soyoil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil,rapeseed oil, canola oil, safflower oil, sunflower oil, flax seed oil,palm oil, palm kernel oil, coconut oil, babassu oil, shea butter, mangobutter, cocoa butter, wheat germ oil, or rice bran oil; or margarine.

In some embodiments, a fat can be triglycerides, monoglycerides,diglycerides, sphingosides, glycolipids, lecithin, lysolecithin,phospholipids such as phosphatidic acids, lysophosphatidic acids,phosphatidyl cholines, phosphatidyl inositols, phosphatidylethanolamines, or phosphatidyl serines; sphingolipids such assphingomyelins or ceramides; sterols such as stigmasterol, sitosterol,campesterol, brassicasterol, sitostanol, campestanol, ergosterol,zymosterol, fecosterol, dinosterol, lanosterol, cholesterol, orepisterol; free fatty acids such as palmitoleic acid, palmitic acid,myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid,caprylic acid, pelargonic acid, undecanoic acid, linoleic acid (C18:2),eicosanoic acid (C22:0), arachidonic acid (C20:4), eicosapentanoic acid(C20:5), docosapentaenoic acid (C22:5), docosahexanoic acid (C22:6),erucic acid (C22:1), conjugated linoleic acid, linolenic acid (C18:3),oleic acid (C18:1), elaidic acid (trans isomer of oleic acid),trans-vaccenic acid (C18:1 trans 11), or conjugated oleic acid; oresters of such fatty acids, including monoacylglyceride esters,diacylglyceride esters, and triacylglyceride esters of such fatty acids.

A fat can comprise phospholipids, sterols or lipids. Phospholipids cancomprise a plurality of amphipathic molecules comprising fatty acids(e.g., see above), glycerol and polar groups. In some embodiments, thepolar groups are, for example, choline, ethanolamine, serine, phosphate,glycerol-3-phosphate, inositol or inositol phosphates. In someembodiments, the lipids are, for example, sphingolipids, ceramides,sphingomyelins, cerebrosides, gangliosides, ether lipids, plasmalogensor pegylated lipids.

In some embodiments, an emulsion is prepared by preparing a solutioncomprising the one or more proteins, admixing said solution with one ormore fats, thereby creating said emulsion. The ratio of protein solutionto fats can be about 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1,or 10:1. The ratio of protein solution to fats can be about 10:1-1:2,1:4-2:1, 1:1-4:1, or 2:1-10:1. The emulsion can be used as a non-dairymilk for the preparation of a non-dairy cheese. By way of example only,0%-50% fat can be added to a protein solution by weight/weight orweight/volume.

Flavor compounds can be generated by microbes in the non-animal derivedmaterial used for producing many different non-dairy products describedherein, including cheese replicas. The methods of flavoring generallyinclude contacting a non-dairy milk or protein solution with one or moremicrobes, and preparing a cultured non-dairy product from the non-dairymilk. Microbes such as bacteria, yeast, or mold can be used to create aproduct with a desired flavor profile or be used as a component of theflavor in a product, as bacteria can create desirable flavors (e.g.,buttery, creamy, dairy, or cheesy) in a neutral, planty, or beanyproduct.

Exemplary non-dairy milks are described herein. Any of the non-dairycheese milks or combinations thereof may be contacted with one or moremicrobes (e.g., a controlled amount of bacteria) to control the flavorof a resulting cultured non-dairy product such as a cheese replica. Insome embodiments, the microbes can be selected from bacteria, yeast, ormolds. In some embodiments, the bacteria can comprise mesophilic and/orthermophilic bacteria. In some embodiments, the bacteria can comprisebacteria from a commercial starter. Exemplary commercial starters aredescribed herein.

Flavor production in the replicas can be controlled by the use of one ormore microbes e.g., one or more bacteria, yeast, or molds, including butnot limited to flavor production in the replicas can be controlled bythe use of one or more microbes e.g., one or more bacteria, yeast, ormolds, including but not limited to Lactococcus species such asLactococcus lactis lactis (LLL, used alone or as a component ofcommercial mix MA11), Lactococcus lactis cremoris (LLC, used alone or asa component of commercial mix MA11), or Lactococcus lactis biovardiacetylactis (LLBD, often used as commercial culture MD88), aLactobacillus species such as Lactobacillus delbrueckii lactis,Lactobacillus delbrueckii bulgaricus, Lactobacillus helveticus,Lactobacillus plantarum, Lactobacillus casei, or Lactobacillusrhamnosus, a Leuconostocaceae species such as Leuconostoc mesenteroidescremoris (LM), a Streptococcus species such as Streptococcusthermophiles (ST, often used as commercial culture TA61) a Pediococcusspecies such as Pediococcus pentosaceus, a Clostridium species such asClostridium butyricum, a Staphylococcus species such as Staphylococcusxylosus (SX), a Brevibacterium species such as Brevibacterium linens, aPropioniibacteria species, a Penicillium species such as Penicilliumcandidum, Penicillium camemberti, or Penicillium roqueforti, aDebaryomyces species such as Debaryomyces hansenii, a Geotrichum speciessuch as Geotrichum candidum, a Corynebacteria species, a Verticilliumspecies such as Verticillium lecanii, a Kluyveromyces species such asKluyveromyces lactis, a Saccharomyces species such as Saccharomycescerevisiae, a Candida species such as Candida jefer or Candida utilis, aRhodosporidum species such as Rhodosporidum infirmominiatum, aMicrococcus species, a Halomonas species, a Psychrobacter species. Insome embodiments, lactic acid bacteria such as Lactobacillus,Leuconostoc, Pediococcus, Lactococcus, or Streptococcus are used. Insome embodiments, the bacteria do not comprise Lactobacciliusacidophilus strains. In some embodiments, a yeast such as Saccharomycescerevisiae, Kluveromyces lactis and/or Debaromyces hansenii can be used.In some embodiments, a mold can be Penicillium candidum, Penicilliumcamemberti, Penicillium roqueforti, Geotrichum candidum, or acombination thereof.

In some embodiments, one or more of the follow microbes are used:Pediococcus pentosaceus, Clostridium butyricum, Lactobacillusdelbrueckii lactis, Lactobacillus delbrueckii bulgaricus, Lactobacillushelveticus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillusrhamnosus, Staphylococcus xylosus, and Brevibacterium linens.

In some embodiments, a non-dairy cheese source can be cultured with oneor more microbes (e.g., bacteria, yeast, or mold alone), or incombination with two or more microbes (e.g., two different bacteria, twodifferent yeast, two different molds, a bacteria and a yeast, a bacteriaand a mold, or a yeast and a mold). When two or more microbes are used,the microbes can be co-cultured or sequentially cultured, i.e., onemicrobe can be cultured for a length of time before adding anothermicrobe. Particular good combinations for flavor generation in replicasare pre-culturing with SX, followed by either TA61 or MD88, or MD88co-cultured with MA11.

Growth conditions of microbes also can control flavor generation inreplicas. The temperature of microbes growth ranging from 4° C. to 45°C. can control the amount and type of flavor compounds produced inreplicas. The amount of aeration by shaking (e.g., 0 to 300 rpm) canchange the flavor productions of many different bacteria in non-dairymedia. Greater aeration during culturing by either SX, TA61, or MD88 cangenerate more desired cheese and buttery compounds. Aeration can alsodecrease some undesired flavor compounds. Desired cheese compounds suchas 2-heptanone can increase when SX, MD88, or TA61 are cultured withaeration. MD88's production of hexanoic methyl ester in cheese replicascan also be modulated by aeration. An increase in aeration of SX duringculturing in soymilk can increase 3-methyl and 2-methyl butanoic acidproduction and can decrease the amounts of undesirable aroma compoundssuch as 2-ethyl furan or 2-pentyl furan in cheese replicas.

The amount of time the one or more microbes is cultured also canmodulate the amount and types of flavor compounds. In some embodiments,culturing can range from 1 hour to multiple days. In some embodiments,one or more microbes and the non-dairy milk are incubated together for alength of time ranging from 1 min-60 minutes, 0.5-5 hours, 3-10 hours,6-15 hours, 10-20 hours, or more than 20 hours. In some embodiments,most buttery compounds are created within the first 10 hours, whileadditional cheese compounds can be formed in 24-48 hours or more hours.Butyrolactone, a creamy, milky note compound can be created in non-dairymedia (e.g., a non-dairy cheese source or a milk replica) by MD88 andMA11 only after 20 hours of culturing in soymilk.

In some embodiments, the one or more microbes also can be added atdifferent inoculums, e.g., 10²-10⁹ cfu/mL or even greater. The phase ofgrowth (i.e, stationary phase versus exponential phase) and the celldensity of the bacterial culture can affect the flavor compound profileof the medium. Higher inocula of a starter culture can protect thereplica from unwanted microbial contamination (e.g., bacterialcontamination). Therefore, an inoculum of 10⁶-10⁹ cfu/mL is usuallyused.

Flavor production by the one or more microbes also can be modulated bydirecting the metabolic pathways, e.g., by modulating their nitrogensource, carbon source, additional available nutrients, and growthconditions.

In some embodiments, the one or more microbes, the non-dairy cheesesource, and the one or more optional components that can be used toalter flavor (e.g., sugars, fats, carbohydrates, vitamins, organicacids, nucleotides, or food products) are incubated together for asufficient period of time to achieve a desired pH. The pH can range frompH 3-5, 4-6, or 4.3-5.7. The desired pH can be pH 6 or lower, pH 5 orlower, or pH 4 or lower. Culturing the material by bacteria in somecases decreases the pH to 6.5, 6, 5.5, 5, 4.5, 4, or 3.5, while in othercases, flavors are generated with no change in pH. Culturing withLactococcus, Lactobacillus, Leuconostoc, Pediococcus and/orStreptococcus generally results in a decrease in pH with most startingmaterial, while culturing with Staphylococcus, Brevibacterium, and/orClostridium generally has little or no effect on the pH.

In some embodiments, one or more enzymes can be used alone or incombination any one the culturing methods and additives described tohelp modulate the flavor, texture, and/or melting profile, comprisingcontacting a non-dairy cheese source with one or more enzymes. In someembodiments, the one or more enzymes can be added before solidification,after solidification but before the whey is drained, or after whey isdrained. Surprisingly, adding trace amounts of one or more enzymes(e.g., proteases, lipases, and/or amylases) can enhance the texture,flavor, and/or meltability of the resulting non-dairy cheese replica, asdetermined by blind taste test or by the detection of volatile odorantsby, e.g., GCMS. Using such enzymes can also impact flavor production bymicrobial cultures (e.g., when soymilk is pre-treated with amylases,TA61 produces much more diacetyl).

In some embodiments, the enzyme is aspartic protease.

In specific embodiments, the protease is papain, bromelain, AO protease,figin, rennet, protease type XXI from Streptomyces griseus, a proteasefrom Bacillus licheniformis, a protease from Aspergillus oryzae, aprotease from Bacillus amyloliquefaciens, a protease from Aspergillussaitoi, a thermolysin from Bacillus thermoproteolyticus rokko,Subtilisin A, protease type X, or a fungal protease type XIII.

In some embodiments, the enzyme is a lipase.

The added enzyme can account for 0.00001-0.005%, 0.001-0.01%, 0.01-0.1%,0.05-1%, 0.1-2%, or 0.5-5% of the non-dairy cheese source by weight orvolume. In some embodiments, the added enzyme can account for0.00001-0.1% of the non-dairy cheese source by weight or volume.

In some embodiments, the protease is papain. In some embodiments,0.001-0.01% of papain is added to the non-dairy cheese source. In someembodiments, a protein solution with added protease is solidified by aheat/cool method. In some embodiments, addition of papain improves thesoftness and creaminess of the resulting cheese replica.

A method can comprise adding one or more fats to the non-dairy cheesesource to create an emulsion.

By way of example only, some non-dairy cheese replicas can be preparedby adding 0%-50% fat to a non-dairy cheese source to create an emulsion,then solidifying the emulsion by protein denaturation, e.g., by heating.In some embodiments, one or more fats are added before solidifying, orafter solidifying. In some embodiments, the one or more fats are addedafter solidifying and after draining the whey. By way of other exampleonly, some cheese replicas made from protein denaturation have 0% to 50%fat added after solidification by denaturation, or 0% to 50% fat addedafter draining the whey. In some embodiments, after formation of a gel,either by protein denaturation or crosslinking, whey can be drained toincrease the total fat in the cheese replica, further draining and agingthe cheese can reduce the moisture content to increase the total fat ofthe cheese replica.

In some embodiments, the addition of 5-20% unsaturated fats to enzymecrosslinked gels can increase the firmness of the gel.

In some embodiments, addition of saturated fats from 5%-50% can increasethe firmness of the cheese replicas.

In another aspect, the disclosure provides cheese replicas and methodsof making the same. In some embodiments, the method comprisessolidifying a non-dairy cheese source (e.g., a non-dairy milk) (e.g., byforming a gel). In some embodiments, the non-dairy milk is capable ofretaining a shape after said solidifying. There are many ways in whichthe non-dairy cheese source can be solidified, including using enzymes,heat denature, forming cold gels, forming coacervate, liquid separation,acids, change in ionic strength, high pressure processing, solvents,chaotropic agents, or disulfide bond reducers as described in thissection.

Enzymes (or chemicals) can be used to crosslink non-animal (e.g., plantbased) proteins or non-dairy cheese sources, with or without emulsifiedfats or oils, sugars, and cultures. The resulting cross-linked cheesereplicas can have bacteria cultures added or not, and the timing ofaddition can be either before or after the crosslinking step. In someembodiments, solidifying involves a process of cross-linking components(e.g., polypeptides, also referred to as proteins herein) in thenon-dairy cheese source. In some embodiments, cross-linking comprisescontacting the non-dairy cheese source with a cross-linking enzyme,thereby creating crosslinks between polypeptide chains. In someembodiments, a crosslinking enzyme can be a transglutaminase,tyrosinase, lipoxygenase, protein disulfide reductase, protein disulfideisomerase, sulfhydryl oxidase, peroxidase, hexose oxidase, lysyloxidase, or amine oxidase.

In some embodiments, the cross-linking enzyme is a transglutaminase.Transglutaminases are a family of enzymes that catalyze the formation ofa covalent bond between a free amine and the gamma-carboxyl group ofglutamine thereby linking proteins together. For example,transglutaminases catalyze crosslinking of e.g., lysine in a protein orpeptide and the gamma-carboxamide group of a protein- orpeptide-glutamine residue. The covalent bonds formed by transglutaminasecan exhibit high resistance to proteolytic degradation.

Many types of transglutaminase can be used in various embodiments of theinvention. Acceptable transglutaminases for crosslinking include, butare not limited to, Streptoverticillium mobaraense transglutaminase, anenzyme that is similar to a transglutaminase from Streptoverticilliummobaraense, other microbial transglutaminases, transglutaminasesproduced by genetically engineered bacteria, fungi or algae, Factor XIII(fibrin-stabilizing factor), Keratinocyte transglutaminase (TGM1),Tissue transglutaminase (TGM2), Epidermal transglutaminase (TGM3),Prostate transglutaminase (TGM4), TGM X (TGM5), TGM Y (TGM6), TGM Z(TGM7), or a lysyl oxidase.

The timing of adding the cultures, the type of cultures, and amount ofcultures can change the pH of the emulsion, and therefore the activityof transglutaminase and the final texture of the cheese. In addition,changing the pH of the solution with the addition of acid or base, andoverall buffering capacity of the emulsion can alter the crosslinkingability and the final texture of the cheese-replica.

In some embodiments, the present invention provides for a compositioncomprising a non-dairy milk and a Streptoverticillium mobaraensetransglutaminase, an enzyme is similar to a transglutaminase fromStreptoverticillium mobaraense, other microbial transglutaminases,transglutaminases produced by genetically engineered bacteria, fungi oralgae, Factor XIII (fibrin-stabilizing factor), Keratinocytetransglutaminase (TGM1), Tissue transglutaminase (TGM2), Epidermaltransglutaminase (TGM3), Prostate transglutaminase (TGM4), TGM X (TGM5),TGM Y (TGM6) and/or TGM Z (TGM7). In some embodiments the enzyme usedfor cross-linking is not Factor XIII (fibrin-stabilizing factor),Keratinocyte transglutaminase (TGM1), Tissue transglutaminase (TGM2),Epidermal transglutaminase (TGM3), Prostate transglutaminase (TGM4), TGMX (TGM5), TGM Y (TGM6), TGM Z (TGM7), or lysyl oxidase.

Transglutaminases can be produced by Streptoverticillium mobaraensefermentation in commercial quantities or extracted from animal tissues.Additionally, a transglutaminase (TGM) of the present disclosure can beisolated from bacteria or fungi, expressed in bacteria or fungi from asynthetic or cloned gene. In some particular embodiments, atransglutaminase is obtained from a commercial source, for example inthe form of Activa™ from Ajinomoto Food Ingredients LLC.

In some embodiments, a transglutaminase is added at an amount between0.0000001-0.001%, 0.0001-0.1%, 0.001-0.05%, 0.1-2%, 0.5-4%, or greaterthan 4% by weight/volume. In some embodiments, a transglutaminase isadded at amounts greater than 0.1% and up to 10%.

In some embodiments, cross-linking by a transglutaminase can be done attemperatures ranging from 10-30° C., 20-60° C., 30-70° C., or 50-100° C.Transglutaminase cross-linking can occur for 10 minutes-24 hours.

In some embodiments, between 0.1 and 20 units (U) of transglutaminase isadded per 1 mL of non-dairy milk. In some embodiments about 0.1, 0.5, 1,1.5, 2, 2.5, 3, 5, 7, 10, 15, or 20 U of transglutaminase is added per 1mL of non-dairy milk. In some embodiments after the transglutaminase isadded, a heated incubation occurs, for example in a 100° F. water bath.The heated incubation can be at a temperature optimized for the enzymefunction. In some embodiments the temperature is about 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120 or 125° F. In some embodiments,enzymatic cross-linking does not comprise contacting the non-dairycheese source with glutaminase and transglutaminase. Transglutaminasecrosslinking has been done at room temperature, and up to 65° C., for 10minutes to 24 hours.

In some embodiments, solidifying comprises inducing proteindenaturation. In some embodiments, denaturation is induced by heatingthe mixture, followed by cooling the mixture. In some embodiments,denaturation is induced by heating the mixture to a temperature between30-35, 32-40, 37-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, 70-80,75-85, 80-95, 90-100° C., or above 100° C. In some embodiments,denaturation is induced by heating the mixture for about 10-20, 15-30,25-40, 30-50, 40-70 seconds or about 1-3, 2-5, 3-8, or 5-20 minutes. Insome embodiments, the mixture is allowed to cool after heating. Forexample, proteins (e.g., a protein composition as disclosed herein, acommercially available protein, a protein purified by any method knownin the art, purified or fractionated plant proteins such as from peas,moong, soy, RuBisCO, or a combination thereof), preferably atconcentrations >1%, can be homogenized with oils (such as canola oil,sunflower oil, palm oil or oil bodies from seeds such as sunflower) at0.1-60% concentration. The emulsion can be subjected to a heat-coolcycle wherein it is heated to a temperature of 45-100° C. for 5-60minutes and then cooled to less than 30° C. (e.g., 20-25° C.). Theresulting gel can be incubated at a temperature <30° C., preferably for2-16 hours and then drained through cheesecloth. The drained curds areready to be shaped and aged or processed further by heating or pressing.

Acids, change in ionic strength, high pressure processing, solvents,chaotropic agents, or disulfide bond reducers can be used to denaturethe proteins in the non-dairy cheese source. In some embodiments, ureais added to the non-dairy cheese source to form curds.

In some embodiments, solidifying results in the formation of solid curdsand whey (resulting liquid that remains after curd is formed). In someembodiments, the curds are separated from the whey.

In some embodiments, solidifying comprises a combination of two or moremethods. For example, solidifying can include crosslinking proteins anddenaturation by heating followed by cooling. For example, a cold set gelcan be cross-linked with transglutaminase to yield firmer gels orcombined with other proteins such as soy, pea-legumins, pea-albumins,crude protein fraction from chick peas and lentils or materials (forexample, fats or pea protein coacervates) to increase firmness and/ormeltability.

In some embodiments, a non-dairy cheese source can be subjected to ashearing force during said solidifying. Said shear force can be used tocause protein components in said non-dairy cheese source to align,forming anisotropic fibers. Said formation of anisotropic fibers can beuseful in creating a stretch cheese.

In another aspect, the disclosure provides methods for flavoringcultured non-dairy products, including sour cream, crème fraiche,yogurt, or cheese replicas. In some embodiments, the method comprisescomparing a flavor note profile of a test non-dairy product with one ormore flavor additives and/or one or more individual microbial strainsdescribed herein to a flavor note profile of a control non-dairy productwithout the additives and/or individual microbial strain. The textureand flavor profile of a non-dairy product (e.g. cheese replica) can beascertained by any method known in the art or described herein.Exemplary methods of ascertaining flavor and texture can be by a tastetest, e.g., a blind taste test, or using gas chromatography-massspectrometry (GCMS).

GCMS is a method that combines the features of gas-liquid chromatographyand mass spectrometry to identify different substances within a testsample. GCMS can, in some embodiments, be used to evaluate theproperties of a dairy cheese and a cheese replica. For example volatilechemicals can be detected from the head space around a dairy cheese or acheese replica. These chemicals can be identified using GCMS. A profileof the volatile chemicals in the headspace around cheese is therebycreated. In some embodiments, each peak of the GCMS can be furtherevaluated. For instance, a human could rate the experience of smellingthe chemical responsible for a certain peak. This information could beused to further refine the profile. GCMS could then be used to evaluatethe properties of the cheese replicas. The GCMS could be used to refinethe cheese replica. In some embodiments the cheese replica has a GCMSprofile similar to that of dairy cheese. In some embodiments the cheesereplica has a GCMS profile identical to that of dairy cheese.

A flavor profile of a diary replica can be characterized by the presenceand/or intensity of one or more flavor notes. Exemplary flavor notesinclude, but are not limited to butteriness, fruitiness, nuttiness,dairy, milky, cheesy, fatty, fruity, pineapple, waxy, buttery, tonka,dark fruit, citrus, sour, banana-like, sweet, bitter, musty, floral,goaty, sweaty, woody, earthly, mushroom, malty, spicy, pear, green,balsamic, pungent, oily, rose, fatty, butterscotch, orange, pine,carnation, melon, pineapple, vanilla, garlic, herbaceous, woody,cinnamon, rue, yogurt, peach, vanilla, hawthorn, and herbaceous. Theflavor notes may be associated with the release of one or more volatilecompounds. The flavor profile can be characterized by the absence orreduction in the intensity of one or more flavor notes. Exemplary flavornotes include: planty, beany, soy, green, vegetable, nutty, dirty, andsour.

Exemplary volatile compounds include, e.g., gamma-nonanoic lactone,gamma-undecalactone, gamma-decalactone, delta-tetradecalactone, S-methylthiopropionate, delta-tridecalactone, delta-tetradecalactone,δ-tetradecalactone, butyl butyryllactate, 2,3-hexandione, methylhexanoate, butyrolactone, propanoic acid, 2-methyl propanoic acid,methyl isobutyl ketone, gamma octalactone, delta octalactone, gammanonalactone, 5-hydroxy-4-octanone, 2-ethyl-1-hexanol, octane, ethanol,2,3-butanedione, 2 heptanone, 1-butanol, acetoin, butanoic acid,nonanal, acetic acid, 1,3 butanediol, methyl-3-buten-1-ol, methanol,hexanol, dimethyl-benzene, ethyl-benzene, indole, limonene, toluene,acetophenone, pentan-2,3-dione, 2-pentanone, 2-heptanone, 2-nonanone,acetone, butanone, 2-methylpropionic acid, butanoic acid,2-methylbutanoic acid, 3-methylbutanoic acid, pentanoic acid,4-methylpentanoic acid, hexanoic acid, octanoic acid, decanoic acid,undecanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid,octadecanoic acid, oleic acid, linoleic acid, linolenic acid, propanol,butanol, pentanol, hexanol, heptanol, octanol, propan-2-ol, butan-2-ol,pentan-2-ol, hexan-2-ol, heptan-2-ol, nonan-2-ol, undecan-2-ol,octen-3-ol, octa-1,5-dien-3-ol, 3-methyl-2-cyclohexenol,2-methylpropanol, 2-methylbutanol, 3-methylbutanol, 3-methylpentanol,phenylmethanol, 2-phenylethanol, 2-phenyl-ethan-2-ol, propan-2-one,butan-2-one, pentan-2-one, hexan-2-one, heptan-2-one, octan-2-one,nonan-2-one, decan-2-one, undecan-2-one, dodecan-2-one, tridecan-2-one,pentadeca-2-one, pentan-3-one, octan-3-one, 3-methylpentan-2-one,4-methylpentan-2-one, methylhexan-2-one, hydroxypropan-2-one,hept-5-en-2-one, 4-methylpent-3-en-2-one, octen-3-one,octa-1,5-dien-3-one, nonen-2-one, undecen-2-one, methylfuryl ketone,phenylpropan-2-one, propiophenone, methyl butanoate, methyl hexanoate,methyl octanoate, methyl decanoate, methyl tetradecanoate, methylhexadecanoate, methyl cinnamate, ethyl formate, ethyl acetate, ethylpropanoate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyldecanoate, ethyl dodecanoate, ethyl tetradecanoate, ethyl-3-methylbutanoate, propyl acetate, propyl butanoate, butyl formate, butylacetate, amyl acetate, isoamyl formate, isoamyl acetate, isoamylpropanoate, isoamyl butanoate, diethyl phthalate, dimethyl phthalate,2-phenylethyl acetate, 2-phenylethyl propanoate, 2-phenylethylbutanoate, 3-methylthiopropanol, methanethiol, hydrogen sulfide,dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide,methylethyl disulfide, diethyl disulfide, 2,4-dithiapentane, methional,3-methylthio-2,4-dithiapentane, 2,4,5-trithiahexane,1,1-bis-methylmercaptodisulfide, methanethiol acetate, methylthiopropanoate, methyl thiobenzoate, thiophen-2-aldehyde, methylindole,p-ethylphenol, p-cresol, acetaldehyde, butanal, 2-methylbutanal,3-methylbutanal, 2-methylpropanal, hexanal, heptanal, nonanal,2-methylbuten-2-al, benzaldehyde, 3-methylheptyl acetate, 1-butanol,1-butanol, 3-methyl, 1-heptanol, formic acid, 1-hexanol-2, ethyl,1-octanol, 2-butanone, 2-hepten-1-ol, 2-hexanone, heptanal,2-octen-1-ol, 1-octen-3-ol, 2-pentanone, 2,3-butanedione, 3-buten-1-ol,5-Hepten-2-one, octane, ethanol, 2,3-butanedione, 2 heptanone,1-butanol, butanoic acid, nonanal, acetic acid, 1,3 butanediol,methyl-3-buten phenylethyl alcohol, toluene, 1-pentanol, 3-octene-1-ol,2 octene-1-ol, 2-undecanone, 1-octanol, Benzaldehyde, 1-heptanol,2-heptanone, 4-methyl-2-nonanone, 2-methyl-2-nonanol, 1-hexanol,2-methyl 2-propanol, Ethanol, 3 methyl 1-butanol, 1-hexanol, 2-methyl2-nonanol, 2-nonanone, 2-heptanone, 4-methyl, 1-heptanol, 1-octanol, 2octene-1-ol, 3-octene-1-ol, 1-octanol, 1-heptanol, 2-heptanone,4-methyl-2-nonanone, 2-dodecanol, 2-dodecanone, 3-decene 1-01 acetate,benzyl alcohol, phenylethyl alcohol, 2-methoxy 4-vinylphenol, 3-decene1-ol acetate, 2-dodecanone, 2-dodecanol, or 2-methoxy 4-vinylphenol.

In some embodiments, improved flavors are due to the decreased levels ofvolatile flavor compounds, such as, e.g., 1-Hexanol; 2-Butylfuran;2-methyl-2-Pentenal; 3-Octanone; Ethyl-Acetate; 2-Ethyl-Furan;2-pentyl-Furan; Pyrazine; 1-Decanol; Acetophenone; 1-Nonanol;2,5-Dimethyl-Pyrazine; Dodecanal; Benzeneacetaldehyde; Nonanal;Butyrolactone; Octanal; 2-Decanone; Hexanal; 2-Nonanone; Benzaldehyde;Heptanal; 2-Octanone; Furfural; 2-Heptanone; Pentanal.

In some embodiments, the method further comprises preparing a culturednon-dairy product such as a cheese replica, yogurt, sour cream, or crèmefraiche with a controlled flavor profile, by the controlled addition ofdefined combinations of flavor additives, described herein, to thenon-dairy e source at any time point of the replica making process.Exemplary additives and specific combinations are described herein.

Exemplary Embodiments

-   Embodiment 1 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate to obtain a filtered lysate;    -   d) concentrating the filtered lysate to obtain a protein        composition; and    -   e) optionally pasteurizing the protein composition of protein to        obtain a pasteurized protein composition,    -   wherein steps a), b), c), and d) independently, are performed at        a pH between about 8.5 and about 12.0.-   Embodiment 2 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) concentrating the clarified lysate to obtain a protein        composition; and    -   d) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), c), and d) independently, are performed at        a pH between about 8.5 and about 12.0.-   Embodiment 3 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate to obtain a protein        composition; and    -   d) optionally pasteurizing the protein composition, to obtain a        pasteurized protein composition    -   wherein steps a), b), c), and d) independently, are performed at        a pH between about 8.5 and about 12.0.-   Embodiment 4 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate using microfiltration to        obtain a first filtered lysate;    -   d) filtering the first filtered lysate using diafiltration to        obtain a second filtered lysate;    -   e) filtering the second filtered lysate using ultrafiltration to        obtain a third filtered lysate;    -   f) filtering the third filtered lysate using diafiltration to        obtain a protein composition; and    -   g) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), c), d), e), f), and g) independently, are        performed at a pH between about 8.5 and about 12.0.-   Embodiment 5 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate using microfiltration to        obtain a first filtered lysate;    -   d) filtering the first filtered lysate using diafiltration to        obtain a protein composition; and    -   e) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), c), d), and e) independently, are        performed at a pH between about 8.5 and about 12.0.-   Embodiment 6 is the method of any one of embodiments 1-5, wherein    filtering comprises microfiltration.-   Embodiment 7 is the method of any one of embodiments 1-6, wherein    filtering comprises ultrafiltration.-   Embodiment 8 is the method of any one of embodiments 1-7, wherein    filtering comprises diafiltration-   Embodiment 9 is the method of embodiment 8, where diafiltration is    performed for at least two diavolumes.-   Embodiment 10 is the method of any one of embodiments 1-9, wherein    the plurality of cells comprises microbial cells.-   Embodiment 11 is the method of any one of embodiments 1-10, wherein    the plurality of cells comprises fungal cells.-   Embodiment 12 is the method of embodiment 11, wherein the fungal    cells are selected from the group consisting of Saccharomyces,    Pichia, Candida, Hansenula, Torulopsis, Kluyveromyces, Yarrowia, and    Fusarium cells.-   Embodiment 13 is the method of embodiment 11, wherein the fungal    cells are selected from the group consisting of Saccharomyces    cerevisiae, Pichia pastoris, Candida boidinii, Hansenula polymorpha,    Kluyveromyces lactis, Yarrowia lipolytica, and Fusarium venenatum.-   Embodiment 14 is the method of any one of embodiments 1-13, wherein    the plurality of cells comprises bacterial cells.-   Embodiment 15 is the method of embodiment 14, wherein the bacterial    cells are selected from the group consisting of Bacillus,    Escherichia, Lactobacillus, Corynebacterium, Pseudomonas, and    Methanococcus.-   Embodiment 16 is the method of embodiment 14, wherein the bacterial    cells are selected from the group consisting of Escherichia coli,    Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum,    Pseudomonas fluorescens, and Methanococcus maripaludis.-   Embodiment 17 is the method of any one of embodiments 1-16, wherein    the aqueous suspension of the plurality of cells comprises from    about 2% to about 25% dry solids.-   Embodiment 18 is the method of any one of embodiments 1-17, further    comprising washing the aqueous suspension of the plurality of cells    at a pH between about 8.5 and about 12.0 before step a).-   Embodiment 19 is the method of any one of embodiments 1-18, wherein    the lysing step is performed at a temperature between about 4° C.    and about 15° C.-   Embodiment 20 is the method of any one of embodiments 1-19, wherein    the lysing step is performed biochemically.-   Embodiment 21 is the method of any one of embodiments 1-20, wherein    the lysing step is performed chemically.-   Embodiment 22 is the method of any one of embodiments 1-21, wherein    the lysing step is performed mechanically.-   Embodiment 23 is the method of any one of embodiments 1-22, wherein    the lysing step is performed a pH between about 9.0 and about 12.0.-   Embodiment 24 is the method of embodiment 22, wherein the lysing    step is performed at a pH between about 9.0 and about 10.0.-   Embodiment 25 is the method of embodiment 22, wherein the lysing    step is performed at a pH between about 10.0 and about 11.0.-   Embodiment 26 is the method of embodiment 22, wherein the lysing    step is performed at a pH between about 11.0 and about 12.0.-   Embodiment 27 is the method of any one of embodiments 1-26 wherein    the clarifying step is performed, optionally in the presence of one    or more flocculants, at a pH between about 9.0 and about 12.0.-   Embodiment 28 is the method of embodiment 27, wherein the clarifying    step is performed at a pH between about 9.0 and about 10.0.-   Embodiment 29 is the method of embodiment 27, wherein the clarifying    step is performed at a pH between about 10.0 and about 11.0.-   Embodiment 30 is the method of embodiment 27, wherein the clarifying    step is performed at a pH between about 11.0 and about 12.0.-   Embodiment 31 is the method of any one of embodiments 1-30, wherein    clarifying step is performed by centrifugation to less than about    20% dry solids.-   Embodiment 32 is the method of any one of embodiments 1-31, wherein    the clarifying step is performed by gravity settling to less than    about 20% dry solids.-   Embodiment 33 is the method of any one of embodiments 1-32, wherein    the clarifying step is performed by diatomaceous earth filtration to    less than about 20% dry solids.-   Embodiment 34 is the method of any one of embodiments 1-33, wherein    the lysate is diluted 1:1 with water or aqueous solution of salt or    buffer before clarifying, wherein the pH is between about 8.5 and    about 12.0.-   Embodiment 35 is the method of any one of embodiments 1-34, wherein    the cell lysate from step a) is clarified in the presence of one or    more flocculants.-   Embodiment 36 is the method of embodiment 35, wherein the one or    more flocculants comprise one or more of alkylamine epichlorohydrin,    polydimethyldiallylammonium chloride, a polyamine, lime, hydrated    lime, ferric chloride, ferric sulfate, ferrous sulfate, aluminum    sulfate, sodium aluminate, aluminum chloride, magnesium carbonate    hydroxide, calcium carbonate, calcium hydroxide, an activated    silicate, a guar gum, a starch, a tannin, sodium alginate,    polyaluminum sulfate, polyaluminum hydroxy chloride, BIO-FLOCK®, and    a synthetic polyelectrolyte.-   Embodiment 37 is the method of embodiment 36, wherein the one or    more flocculants are selected from the group consisting of    alkylamine epichlorohydrin, polydimethyldiallylammonium chloride, a    polyamine, lime, hydrated lime, ferric chloride, ferric sulfate,    ferrous sulfate, aluminum sulfate, sodium aluminate, aluminum    chloride, magnesium carbonate hydroxide, calcium carbonate, calcium    hydroxide, an activated silicate, a guar gum, a starch, a tannin,    sodium alginate, polyaluminum sulfate, polyaluminum hydroxy    chloride, BIO-FLOCK®, and a synthetic polyelectrolyte.-   Embodiment 38 is the method of any one of embodiments 1-37, wherein    the protein composition has a protein content of about 2 mg/mL to    about 250 mg/mL.-   Embodiment 39 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits one or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 40 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits two or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 41 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits three or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 42 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits four or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 43 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits five or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 44 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits six or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 45 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits seven or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 46 is the method of any one of embodiments 1-38, wherein    the protein composition exhibits the characteristics:    -   H₂S is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the protein composition, and H₂S is        detectable in an amount of at least about 0.2 ppm in the        headspace about 24 hours at 25° C. after about 25 mM L-cysteine        is added to the protein composition,    -   the protein composition forms a gel upon heating to 65° C.,    -   the protein composition has a particle size distribution D10,        D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively,    -   the protein composition is least about 80% denatured after about        20 minutes at about 85° C.,    -   the protein composition forms a gel with a storage modulus of at        least about 100 Pa when heated at or above about 85° C. for        about 20 minutes,    -   wherein the protein composition forms a gel between about pH 5.5        and about pH 10.0,    -   the protein composition forms a gel in solutions with ionic        strength below about 0.5 M, wherein the ionic strength is        calculated based on the concentration of non-protein solutes,        and    -   the protein composition has an emulsion activity index of        greater than or equal to about 50 m²/g protein across about pH        4.0 to about pH 8.0.-   Embodiment 47 is the method of any one of embodiments 1-45, wherein    the protein composition comprises at least about 35%, on a dry    weight basis, of compounds larger than 5 kDa.-   Embodiment 48 is the method of any one of embodiments 1-45, wherein    the protein composition comprises at least about 40%, on a dry    weight basis, of compounds larger than 5 kDa.-   Embodiment 49 is the method of any one of embodiments 1-45, wherein    the protein composition comprises at least about 50%, on a dry    weight basis, of compounds larger than 5 kDa.-   Embodiment 50 is the method of any one of embodiments 1-45, wherein    the protein composition comprises at least about 60%, on a dry    weight basis, of compounds larger than 5 kDa.-   Embodiment 51 is the method of any one of embodiments 1-45, wherein    the protein composition comprises at least about 70%, on a dry    weight basis, of compounds larger than 5 kDa.-   Embodiment 52 is the method of any one of embodiments 47-51, wherein    the compounds larger than 5 kDa are compounds larger than 10 kDa.-   Embodiment 53 is the method of any one of embodiments 47-51, wherein    the compounds larger than 5 kDa are compounds larger than 15 kDa.-   Embodiment 54 is the method of any one of embodiments 47-51, wherein    the compounds larger than 5 kDa are compounds larger than 20 kDa.-   Embodiment 55 is the method of any one of embodiments 47-51, wherein    the compounds larger than 5 kDa are compounds larger than 25 kDa.-   Embodiment 56 is the method of any one of embodiments 1-55, further    comprising drying the protein composition.-   Embodiment 57 is the method of embodiment 56, wherein the protein    composition is spray dried.-   Embodiment 58 is the method of embodiment 56, wherein the protein    composition is freeze dried.-   Embodiment 59 is the method of any one of embodiments 1-55, further    comprising pasteurizing the protein composition to obtain a    pasteurized protein composition.-   Embodiment 60 is the method of embodiment 59, wherein the protein    composition is pasteurized by microfiltration.-   Embodiment 61 is the method of embodiment 59, wherein the protein    composition is pasteurized by high-temperature short time    pasteurization.-   Embodiment 62 is the method of embodiment 59, wherein the protein    composition is pasteurized by adding one or more antimicrobials.-   Embodiment 63 is the method of any one of embodiments 59-62, further    comprising drying the pasteurized protein composition.-   Embodiment 64 is the method of embodiment 63, wherein the    pasteurized protein composition is spray dried.-   Embodiment 65 is the method of embodiment 63, wherein the    pasteurized protein composition is freeze dried.-   Embodiment 66 is the method of any one of embodiments 1-65, wherein    the amount of one or more volatile compounds is reduced by at least    about 1.05-fold compared to a corresponding method in which one or    more of the lysing, clarifying, or filtering steps are not performed    at a pH between about 8.5 and about 12.0, wherein the volatile    compound is selected from the group consisting of cysteine,    1-hexanol, 2-butylfuran, 2-methyl-2-pentenal, 3-octanone, ethyl    acetate, 2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol,    acetophenone, 1-nonanol, 2,5-dimethyl-pyrazine, dodecanal,    benzeneacetaldehyde, nonanal, butyrolactone, octanal, 2-decanone,    hexanal, 2-nonanone, benzaldehyde, heptanal, 2-octanone, furfural,    2-heptanone, and pentanal.-   Embodiment 67 is the method of any one of embodiments 1-66, wherein    the protein composition does not comprise one or more compounds    selected from the group consisting of cysteine, 1-hexanol,    2-butylfuran, 2-methyl-2-pentenal, 3-octanone, ethyl acetate,    2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol, acetophenone,    1-nonanol, 2,5-dimethyl-pyrazine, dodecanal, benzeneacetaldehyde,    nonanal, butyrolactone, octanal, 2-decanone, hexanal, 2-nonanone,    benzaldehyde, heptanal, 2-octanone, furfural, 2-heptanone, and    pentanal.-   Embodiment 68 is the method of any one of embodiments 1-67, wherein    at least about 50% of the protein in the protein composition falls    between about 10 kDa and about 200 kDa.-   Embodiment 69 is the method of any one of embodiments 1-3, wherein    filtering the clarified lysate comprises microfiltering the    clarified lysate using a filter having an average pore diameter from    0.2-2.0 μm and/or diafiltering the clarified lysate to produce the    filtered lysate.-   Embodiment 70 is the method of embodiment 69, wherein the    diafiltering comprises using an ultrafiltration membrane system.-   Embodiment 71 is the method of any one of embodiments 1-2, wherein    the filtered lysate from step c), before concentrating, is further    filtered.-   Embodiment 72 is the method of embodiment 71, wherein the filtered    lysate is ultrafiltered using a membrane having from about 10 kDa to    about 30 kDa molecular weight cutoff.-   Embodiment 73 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) filtering the cell lysate to obtain a protein composition;        and    -   c) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), and c) independently, are performed at a        pH between about 8.5 and about 12.0.-   Embodiment 74 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) filtering the cell lysate using microfiltration obtain a        first filtered lysate;    -   c) filtering the first filtered lysate using diafiltration to        obtain a protein composition; and    -   d) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), c), and d) independently, are performed at        a pH between about 8.5 and about 12.0.-   Embodiment 75 is a method for purifying protein from a plurality of    cells, the method comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) filtering the cell lysate using microfiltration to obtain a        protein composition; and    -   c) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), and c) independently, are performed at a        pH between about 8.5 and about 12.0.-   Embodiment 76 is the method of any one of embodiments 73-75, wherein    filtering comprises microfiltration.-   Embodiment 77 is the method of any one of embodiments 73-76, wherein    filtering comprises ultrafiltration.-   Embodiment 78 is the method of any one of embodiments 73-77, wherein    filtering comprises diafiltration-   Embodiment 79 is the method of embodiment 78, where diafiltration is    performed for at least two diavolumes.-   Embodiment 80 is the method of any one of embodiments 73-79, wherein    the plurality of cells comprises microbial cells.-   Embodiment 81 is the method of any one of embodiments 73-80, wherein    the plurality of cells comprises fungal cells.-   Embodiment 82 is the method of embodiment 81, wherein the fungal    cells are selected from the group consisting of Saccharomyces,    Pichia, Candida, Hansenula, Torulopsis, Kluyveromyces, Yarrowia, and    Fusarium cells.-   Embodiment 83 is the method of embodiment 81, wherein the fungal    cells are selected from the group consisting of Saccharomyces    cerevisiae, Pichia pastoris, Candida boidinii, Hansenula polymorpha,    Kluyveromyces lactis, Yarrowia lipolytica, and Fusarium venenatum.-   Embodiment 84 is the method of any one of embodiments 73-83, wherein    the plurality of cells comprises bacterial cells.-   Embodiment 85 is the method of embodiment 84, wherein the bacterial    cells are selected from the group consisting of Bacillus,    Escherichia, Lactobacillus, Corynebacterium, Pseudomonas, and    Methanococcus.-   Embodiment 86 is the method of embodiment 84, wherein the bacterial    cells are selected from the group consisting of Escherichia coli,    Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum,    Pseudomonas fluorescens, and Methanococcus maripaludis.-   Embodiment 87 is the method of any one of embodiments 73-86, wherein    the aqueous suspension of the plurality of cells comprises from    about 2% to about 25% dry solids.-   Embodiment 88 is the method of any one of embodiments 73-87, further    comprising washing the aqueous suspension of the plurality of cells    at a pH between about 8.5 and about 12.0 before step a).-   Embodiment 89 is the method of any one of embodiments 73-88, wherein    the lysing step is performed at a temperature between about 4° C.    and about 15° C.-   Embodiment 90 is the method of any one of embodiments 73-89, wherein    the lysing step is performed biochemically.-   Embodiment 91 is the method of any one of embodiments 73-90, wherein    the lysing step is performed chemically.-   Embodiment 92 is the method of any one of embodiments 73-91, wherein    the lysing step is performed mechanically.-   Embodiment 93 is the method of any one of embodiments 73-92, wherein    the lysing step is performed at a pH between about 9.0 and about    12.0.-   Embodiment 94 is the method of embodiment 93, wherein the lysing    step is performed at a pH between about 9.0 and about 10.0.-   Embodiment 95 is the method of embodiment 93, wherein the lysing    step is performed at a pH between about 10.0 and about 11.0.-   Embodiment 96 is the method of embodiment 93, wherein the lysing    step is performed at a pH between about 11.0 and about 12.0.-   Embodiment 97 is the method of any one of embodiments 73-96, wherein    the lysate is diluted 1:1 with water or aqueous solution of salt or    buffer before filtering, wherein the pH is between about 8.5 and    about 12.0.-   Embodiment 98 is the method of any one of embodiments 73-97, wherein    the protein composition has a protein content of about 2 mg/mL to    about 250 mg/mL.-   Embodiment 99 is the method of any one of embodiments 73-98, wherein    the protein composition exhibits one or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 100 is the method of any one of embodiments 73-98,    wherein the protein composition exhibits two or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 101 is the method of any one of embodiments 73-98,    wherein the protein composition exhibits three or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 fun, respectively;    the protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 102 is the method of any one of embodiments 73-98,    wherein the protein composition exhibits four or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 103 is the method of any one of embodiments 73-98,    wherein the protein composition exhibits five or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 104 is the method of any one of embodiments 73-98,    wherein the protein composition exhibits six or more characteristics    selected from the group consisting of: H₂S is detectable in an    amount of less than about 0.1 ppm when L-cysteine is not added to    the protein composition, and H₂S is detectable in an amount of at    least about 0.2 ppm in the headspace about 24 hours at 25° C. after    about 25 mM L-cysteine is added to the protein composition; the    protein composition forms a gel upon heating to 65° C.; the protein    composition has a particle size distribution D10, D50, and D90 of    less than 0.1 μm, 1.0 μm and 5 μm, respectively; the protein    composition is least about 80% denatured after about 20 minutes at    about 85° C.; the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the protein composition forms a gel    between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 105 is the method of any one of embodiments 73-98,    wherein the protein composition exhibits seven or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and 112S is detectable in    an amount of at least about 0.2 ppm in the headspace about 24 hours    at 25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 106 is the method of any one of embodiments 73-98,    wherein the protein composition exhibits the characteristics:    -   H₂S is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the protein composition, and H₂S is        detectable in an amount of at least about 0.2 ppm in the        headspace about 24 hours at 25° C. after about 25 mM L-cysteine        is added to the protein composition,    -   the protein composition forms a gel upon heating to 65° C.,    -   the protein composition has a particle size distribution D10,        D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively,    -   the protein composition is least about 80% denatured after about        20 minutes at about 85° C.,    -   the protein composition forms a gel with a storage modulus of at        least about 100 Pa when heated at or above about 85° C. for        about 20 minutes,    -   wherein the protein composition forms a gel between about pH 5.5        and about pH 10.0,    -   the protein composition forms a gel in solutions with ionic        strength below about 0.5 M, wherein the ionic strength is        calculated based on the concentration of non-protein solutes,        and    -   the protein composition has an emulsion activity index of        greater than or equal to about 50 m²/g protein across about pH        4.0 to about pH 8.0.-   Embodiment 107 is the method of any one of embodiments 73-106,    wherein the protein composition comprises at least about 35%, on a    dry weight basis, of compounds larger than 5 kDa.-   Embodiment 108 is the method of any one of embodiments 73-106,    wherein the protein composition comprises at least about 40%, on a    dry weight basis, of compounds larger than 5 kDa.-   Embodiment 109 is the method of any one of embodiments 73-106,    wherein the protein composition comprises at least about 50%, on a    dry weight basis, of compounds larger than 5 kDa.-   Embodiment 110 is the method of any one of embodiments 73-106,    wherein the protein composition comprises at least about 60%, on a    dry weight basis, of compounds larger than 5 kDa.-   Embodiment 111 is the method of any one of embodiments 73-106,    wherein the protein composition comprises at least about 70%, on a    dry weight basis, of compounds larger than 5 kDa.-   Embodiment 112 is the method of any one of embodiments 107-111,    wherein the compounds larger than 5 kDa are compounds larger than 10    kDa.-   Embodiment 113 is the method of any one of embodiments 107-111,    wherein the compounds larger than 5 kDa are compounds larger than 15    kDa.-   Embodiment 114 is the method of any one of embodiments 107-111,    wherein the compounds larger than 5 kDa are compounds larger than 20    kDa.-   Embodiment 115 is the method of any one of embodiments 107-111,    wherein the compounds larger than 5 kDa are compounds larger than 25    kDa.-   Embodiment 116 is the method of any one of embodiments 73-115,    further comprising drying the protein composition.-   Embodiment 117 is the method of embodiment 116, wherein the protein    composition is spray dried.-   Embodiment 118 is the method of embodiment 116, wherein the protein    composition is freeze dried.-   Embodiment 119 is the method of any one of embodiments 73-115,    further comprising pasteurizing the protein composition to obtain a    pasteurized protein composition.-   Embodiment 120 is the method of embodiment 119, wherein the protein    composition is pasteurized by microfiltration.-   Embodiment 121 is the method of embodiment 119, wherein protein    composition is pasteurized by high-temperature short time    pasteurization.-   Embodiment 122 is the method of embodiment 119, wherein the protein    composition is pasteurized by adding one or more antimicrobials.-   Embodiment 123 is the method of any one of embodiments 119-122,    further comprising drying the pasteurized protein composition.-   Embodiment 124 is the method of embodiment 123, wherein the    pasteurized protein composition is spray dried.-   Embodiment 125 is the method of embodiment 123, wherein the    pasteurized protein composition is freeze dried.-   Embodiment 126 is the method of any one of embodiments 73-125,    wherein the amount of one or more volatile compounds is reduced by    at least about 1.05-fold compared to a corresponding method in which    one or more of the lysing or filtering steps are not performed at a    pH between about 8.5 and about 12.0, wherein the volatile compound    is selected from the group consisting of cysteine, 1-hexanol,    2-butylfuran, 2-methyl-2-pentenal, 3-octanone, ethyl acetate,    2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol, acetophenone,    1-nonanol, 2,5-dimethyl-pyrazine, dodecanal, benzeneacetaldehyde,    nonanal, butyrolactone, octanal, 2-decanone, hexanal, 2-nonanone,    benzaldehyde, heptanal, 2-octanone, furfural, 2-heptanone, and    pentanal.-   Embodiment 127 is the method of any one of embodiments 73-126,    wherein the protein composition does not comprise one or more    compounds selected from the group consisting of cysteine, 1-hexanol,    2-butylfuran, 2-methyl-2-pentenal, 3-octanone, ethyl acetate,    2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol, acetophenone,    1-nonanol, 2,5-dimethyl-pyrazine, dodecanal, benzeneacetaldehyde,    nonanal, butyrolactone, octanal, 2-decanone, hexanal, 2-nonanone,    benzaldehyde, heptanal, 2-octanone, furfural, 2-heptanone, and    pentanal.-   Embodiment 128 is the method of any one of embodiments 73-127,    wherein at least about 50% of the protein in the protein composition    falls between about 10 kDa and about 200 kDa.-   Embodiment 129 is a protein composition comprising:    -   a plurality of functional proteins,    -   wherein the protein composition comprises at least about 35%, on        a dry weight basis, compounds larger than 5 kDa.-   Embodiment 130 is a protein composition comprising:    -   a plurality of functional proteins,    -   wherein the protein composition has a buffering capacity of less        than about 2.5 mmol NaOH per gram dry solids.-   Embodiment 131 is a protein composition comprising:    -   a plurality of functional proteins,    -   wherein heating a 10% (w/v) suspension of the protein        composition to at least about 95° C. results in a gel with a        storage modulus of at least about 100 Pa.-   Embodiment 132 is the protein composition of any one of embodiments    129-131, wherein H₂S is detectable in an amount of less than about    0.1 ppm in the headspace after about 24 hours at 25° C. when    L-cysteine is not added to 5 mL of a 2% (w/v) suspension of the    protein composition at pH 7.0.-   Embodiment 133 is the protein composition of any one of embodiments    129-132, wherein H₂S is detectable an amount of at least about 0.2    ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to 5 mL of a 2% (w/v) suspension of the protein    composition at pH 7.0.-   Embodiment 134 is a protein composition comprising:    -   a plurality of functional proteins,    -   wherein H₂S is detectable in an amount of less than about 0.1        ppm in the headspace after about 24 hours at 25° C. when        L-cysteine is not added to 5 mL of a 2% (w/v) suspension of the        protein composition at pH 7.0.-   Embodiment 135 is the protein composition of embodiment 134, wherein    H₂S is detectable in an amount of at least about 0.2 ppm in the    headspace about 24 hours at 25° C. after about 25 mM L-cysteine is    added to 5 mL of a 2% (w/v) suspension of the protein composition at    pH 7.0.-   Embodiment 136 is the protein composition of any one of embodiments    129-127, wherein the protein composition comprises at least about    35%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 137 is the protein composition of any one of embodiments    129-136, wherein the protein composition transitions to a gel upon    heating to 65° C.-   Embodiment 138 is the protein composition of any one of embodiments    129-137, wherein the protein composition is at least about 80%    denatured after about 20 minutes at about 85° C.-   Embodiment 139 is the protein composition of any one of embodiments    129-138, wherein the protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes.-   Embodiment 140 is the protein composition of any one of embodiments    129-139, wherein the protein composition can form a gel between    about pH 5.5 and about pH 10.0.-   Embodiment 141 is the protein composition of any one of embodiments    129-140, wherein the protein composition can form a gel in solutions    with ionic strength below about 0.5 M, wherein the ionic strength is    calculated based on the concentration of non-protein solutes.-   Embodiment 142 is the protein composition of any one of embodiments    129-141, wherein the protein composition has a particle size    distribution D10 of less than about 0.1 μm.-   Embodiment 143 is the protein composition of any one of embodiments    129-142, wherein the protein composition has a particle size    distribution D50 of less than about 1.0 μm.-   Embodiment 144 is the protein composition of any one of embodiments    129-143, wherein the protein composition has a particle size    distribution D90 of less than about 5 μm.-   Embodiment 145 is the protein composition of any one of embodiments    129-144, wherein the protein composition has an emulsion activity    index of greater than or equal to about 50 m²/g protein across about    pH 4.0 to about pH 8.0.-   Embodiment 146 is the protein composition of any one of embodiments    129 or 131-145, wherein the protein composition has a buffering    capacity of less than about 2.5 mmol NaOH per gram dry solids-   Embodiment 147 is the protein composition of any one of embodiments    129-146, wherein the protein composition displays activity in one or    more multi-step metabolic pathways.-   Embodiment 148 is the protein composition of any one of embodiments    129-147, wherein the plurality of functional proteins comprises at    least 10 different functional proteins.-   Embodiment 149 is the protein composition of any one of embodiments    129-148, wherein the plurality of functional proteins comprises at    least 20 different functional proteins.-   Embodiment 150 is the protein composition of any one of embodiments    129-149, wherein the plurality of functional proteins comprises at    least 50 different functional proteins.-   Embodiment 151 is the protein composition of any one of embodiments    129-150, wherein the plurality of functional proteins comprises    functional microbial proteins.-   Embodiment 152 is the protein composition of any one of embodiments    129-151, wherein the plurality of functional proteins comprises    functional fungal proteins.-   Embodiment 153 is the protein composition of any one of embodiments    129-152, wherein the plurality of functional proteins comprises    functional bacterial proteins.-   Embodiment 154 is the protein composition of any one of embodiments    129-153, wherein the plurality of functional proteins comprises    functional proteins from Saccharomyces, Pichia, Candida, Hansenula,    Torulopsis, Kluyveromyces, Yarrowia, Aspergillus, Trichoderma, or    Fusarium.-   Embodiment 155 is the protein composition of any one of embodiments    129-154, wherein the plurality of functional proteins comprises    functional proteins from Saccharomyces cerevisiae, Pichia pastoris,    Candida boidinii, Hansenula polymorpha, Kluyveromyces lactis,    Yarrowia lipolytica, or Fusarium venenatum.-   Embodiment 156 is the protein composition of any one of embodiments    129-155, wherein the plurality of functional proteins comprises    functional proteins from Bacillus, Escherichia, Lactobacillus,    Corynebacterium, Pseudomonas, or Methanococcus.-   Embodiment 157 is the protein composition of any one of embodiments    129-156, wherein the plurality of functional proteins comprises    functional proteins from E. coli, Bacillus subtilis, Lactobacillus    lactis, Corynebacterium glutamicum, Pseudomonas fluorescens, or    Methanococcus maripaludis.-   Embodiment 158 is the protein composition of any one of embodiments    129-157, wherein the plurality of functional proteins comprises one    or more heterologous functional proteins.-   Embodiment 159 is the protein composition of any one of embodiments    129 or 136-158, wherein the protein composition comprises at least    about 40%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 160 is the protein composition of any one of embodiments    129 or 136-158, wherein the protein composition comprises at least    about 50%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 161 is the protein composition of any one of embodiments    129 or 136-158, wherein the protein composition comprises at least    about 60%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 162 is the protein composition of any one of embodiments    129 or 136-158, wherein the protein composition comprises at least    about 70%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 163 is the protein composition of any one of embodiments    129 or 136-158, wherein the protein composition comprises at least    about 80%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 164 is the protein composition of any one of embodiments    129 or 136-163, wherein the compounds larger than 5 kDa are    compounds larger than 10 kDa.-   Embodiment 165 is the protein composition of any one of embodiments    129 or 136-163, wherein the compounds larger than 5 kDa are    compounds larger than 15 kDa.-   Embodiment 166 is the protein composition of any one of embodiments    129 or 136-163, wherein the compounds larger than 5 kDa are    compounds larger than 20 kDa.-   Embodiment 167 is the protein composition of any one of embodiments    129 or 136-163, wherein the compounds larger than 5 kDa are    compounds larger than 25 kDa.-   Embodiment 168 is the protein composition of any one of embodiments    129-167, wherein the protein composition does not comprise one or    more compounds selected from the group consisting of cysteine,    1-hexanol, 2-butylfuran, 2-methyl-2-pentenal, 3-octanone, ethyl    acetate, 2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol,    acetophenone, 1-nonanol, 2,5-dimethyl-pyrazine, dodecanal,    benzeneacetaldehyde, nonanal, butyrolactone, octanal, 2-decanone,    hexanal, 2-nonanone, benzaldehyde, heptanal, 2-octanone, furfural,    2-heptanone, and pentanal.-   Embodiment 169 is the protein composition of any one of embodiments    129-168, wherein at least about 50% of the protein in the protein    composition falls between about 10 kDa and about 200 kDa.-   Embodiment 170 is a Saccharomyces cerevisiae protein composition    comprising: a plurality of functional Saccharomyces cerevisiae    proteins,    -   wherein the Saccharomyces cerevisiae protein composition        comprises at least about 35%, on a dry weight basis, compounds        larger than 5 kDa, and    -   wherein the Saccharomyces cerevisiae protein composition        exhibits two or more characteristics selected from the group        consisting of: 112S is detectable in an amount of less than        about 0.1 ppm when L-cysteine is not added to the Saccharomyces        cerevisiae protein composition, and 112S is detectable in an        amount of at least about 0.2 ppm in the headspace about 24 hours        at 25° C. after about 25 mM L-cysteine is added to the        Saccharomyces cerevisiae protein composition; the Saccharomyces        cerevisiae protein composition forms a gel upon heating to 65°        C.; the Saccharomyces cerevisiae protein composition has a        particle size distribution D10, D50, and D90 of less than 0.1        μm, 1.0 μm and 5 μm, respectively; the Saccharomyces cerevisiae        protein composition is least about 80% denatured after about 20        minutes at about 85° C.; the Saccharomyces cerevisiae protein        composition forms a gel with a storage modulus of at least about        100 Pa when heated at or above about 85° C. for about 20        minutes; the Saccharomyces cerevisiae protein composition forms        a gel between about pH 5.5 and about pH 10.0; the Saccharomyces        cerevisiae protein composition forms a gel in solutions with        ionic strength below about 0.5 M, wherein the ionic strength is        calculated based on the concentration of non-protein solutes;        and the Saccharomyces cerevisiae protein composition has an        emulsion activity index of greater than or equal to about 50        m²/g protein across about pH 4.0 to about pH 8.0.-   Embodiment 171 is the Saccharomyces cerevisiae protein composition    of embodiment 170, wherein the Saccharomyces cerevisiae protein    composition exhibits three or more characteristics selected from the    group consisting of: H₂S is detectable in an amount of less than    about 0.1 ppm when L-cysteine is not added to the Saccharomyces    cerevisiae protein composition, and H₂S is detectable in an amount    of at least about 0.2 ppm in the headspace about 24 hours at 25° C.    after about 25 mM L-cysteine is added to the Saccharomyces    cerevisiae protein composition; the Saccharomyces cerevisiae protein    composition forms a gel upon heating to 65° C.; the Saccharomyces    cerevisiae protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    Saccharomyces cerevisiae protein composition is least about 80%    denatured after about 20 minutes at about 85° C.; the Saccharomyces    cerevisiae protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Saccharomyces cerevisiae protein composition forms a    gel between about pH 5.5 and about pH 10.0; the Saccharomyces    cerevisiae protein composition forms a gel in solutions with ionic    strength below about 0.5 M, wherein the ionic strength is calculated    based on the concentration of non-protein solutes; and the    Saccharomyces cerevisiae protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 172 is the Saccharomyces cerevisiae protein composition    of embodiment 170, wherein the Saccharomyces cerevisiae protein    composition exhibits four or more characteristics selected from the    group consisting of: H₂S is detectable in an amount of less than    about 0.1 ppm when L-cysteine is not added to the Saccharomyces    cerevisiae protein composition, and H₂S is detectable in an amount    of at least about 0.2 ppm in the headspace about 24 hours at 25° C.    after about 25 mM L-cysteine is added to the Saccharomyces    cerevisiae protein composition; the Saccharomyces cerevisiae protein    composition forms a gel upon heating to 65° C.; the Saccharomyces    cerevisiae protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    Saccharomyces cerevisiae protein composition is least about 80%    denatured after about 20 minutes at about 85° C.; the Saccharomyces    cerevisiae protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Saccharomyces cerevisiae protein composition forms a    gel between about pH 5.5 and about pH 10.0; the Saccharomyces    cerevisiae protein composition forms a gel in solutions with ionic    strength below about 0.5 M, wherein the ionic strength is calculated    based on the concentration of non-protein solutes; and the    Saccharomyces cerevisiae protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 173 is the Saccharomyces cerevisiae protein composition    of embodiment 170, wherein the Saccharomyces cerevisiae protein    composition exhibits five or more characteristics selected from the    group consisting of: H₂S is detectable in an amount of less than    about 0.1 ppm when L-cysteine is not added to the Saccharomyces    cerevisiae protein composition, and H₂S is detectable in an amount    of at least about 0.2 ppm in the headspace about 24 hours at 25° C.    after about 25 mM L-cysteine is added to the Saccharomyces    cerevisiae protein composition; the Saccharomyces cerevisiae protein    composition forms a gel upon heating to 65° C.; the Saccharomyces    cerevisiae protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    Saccharomyces cerevisiae protein composition is least about 80%    denatured after about 20 minutes at about 85° C.; the Saccharomyces    cerevisiae protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Saccharomyces cerevisiae protein composition forms a    gel between about pH 5.5 and about pH 10.0; the Saccharomyces    cerevisiae protein composition forms a gel in solutions with ionic    strength below about 0.5 M, wherein the ionic strength is calculated    based on the concentration of non-protein solutes; and the    Saccharomyces cerevisiae protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 174 is the Saccharomyces cerevisiae protein composition    of embodiment 170, wherein the Saccharomyces cerevisiae protein    composition exhibits six or more characteristics selected from the    group consisting of: H₂S is detectable in an amount of less than    about 0.1 ppm when L-cysteine is not added to the Saccharomyces    cerevisiae protein composition, and H₂S is detectable in an amount    of at least about 0.2 ppm in the headspace about 24 hours at 25° C.    after about 25 mM L-cysteine is added to the Saccharomyces    cerevisiae protein composition; the Saccharomyces cerevisiae protein    composition forms a gel upon heating to 65° C.; the Saccharomyces    cerevisiae protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    Saccharomyces cerevisiae protein composition is least about 80%    denatured after about 20 minutes at about 85° C.; the Saccharomyces    cerevisiae protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Saccharomyces cerevisiae protein composition forms a    gel between about pH 5.5 and about pH 10.0; the Saccharomyces    cerevisiae protein composition forms a gel in solutions with ionic    strength below about 0.5 M, wherein the ionic strength is calculated    based on the concentration of non-protein solutes; and the    Saccharomyces cerevisiae protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 175 is the Saccharomyces cerevisiae protein composition    of embodiment 170, wherein the Saccharomyces cerevisiae protein    composition exhibits seven or more characteristics selected from the    group consisting of: H₂S is detectable in an amount of less than    about 0.1 ppm when L-cysteine is not added to the Saccharomyces    cerevisiae protein composition, and H₂S is detectable in an amount    of at least about 0.2 ppm in the headspace about 24 hours at 25° C.    after about 25 mM L-cysteine is added to the Saccharomyces    cerevisiae protein composition; the Saccharomyces cerevisiae protein    composition forms a gel upon heating to 65° C.; the Saccharomyces    cerevisiae protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    Saccharomyces cerevisiae protein composition is least about 80%    denatured after about 20 minutes at about 85° C.; the Saccharomyces    cerevisiae protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Saccharomyces cerevisiae protein composition forms a    gel between about pH 5.5 and about pH 10.0; the Saccharomyces    cerevisiae protein composition forms a gel in solutions with ionic    strength below about 0.5 M, wherein the ionic strength is calculated    based on the concentration of non-protein solutes; and the    Saccharomyces cerevisiae protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 176 is the Saccharomyces cerevisiae protein composition    of embodiment 170, wherein the Saccharomyces cerevisiae protein    composition exhibits the characteristics:    -   H₂S is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the Saccharomyces cerevisiae protein        composition, and H₂S is detectable in an amount of at least        about 0.2 ppm in the headspace about 24 hours at 25° C. after        about 25 mM L-cysteine is added to the Saccharomyces cerevisiae        protein composition,    -   wherein the Saccharomyces cerevisiae protein composition forms a        gel upon heating to 65° C.,    -   wherein the Saccharomyces cerevisiae protein composition has a        particle size distribution D10, D50, and D90 of less than 0.1        μm, 1.0 μm and 5 μm, respectively,    -   wherein the Saccharomyces cerevisiae protein composition is        least about 80% denatured after about 20 minutes at about 85°        C.,    -   wherein the Saccharomyces cerevisiae protein composition forms a        gel with a storage modulus of at least about 100 Pa when heated        at or above about 85° C. for about 20 minutes,    -   wherein the Saccharomyces cerevisiae protein composition forms a        gel between about pH 5.5 and about pH 10.0,    -   wherein the Saccharomyces cerevisiae protein composition forms a        gel in solutions with ionic strength below about 0.5 M, wherein        the ionic strength is calculated based on the concentration of        non-protein solutes, and    -   wherein the Saccharomyces cerevisiae protein composition has an        emulsion activity index of greater than or equal to about 50        m²/g protein across about pH 4.0 to about pH 8.0.-   Embodiment 177 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-176, wherein the protein composition    comprises at least about 40%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 178 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-176, wherein the protein composition    comprises at least about 50%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 179 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-176, wherein the protein composition    comprises at least about 60%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 180 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-176, wherein the protein composition    comprises at least about 70%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 181 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-176, wherein the protein composition    comprises at least about 80%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 182 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-181, wherein the compounds larger than    5 kDa are compounds larger than 10 kDa.-   Embodiment 183 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-181, wherein the compounds larger than    5 kDa are compounds larger than 15 kDa.-   Embodiment 184 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-181, wherein the compounds larger than    5 kDa are compounds larger than 20 kDa.-   Embodiment 185 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-181, wherein the compounds larger than    5 kDa are compounds larger than 25 kDa.-   Embodiment 186 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-185, wherein the protein composition    does not comprise one or more compounds selected from the group    consisting of cysteine, 1-hexanol, 2-butylfuran,    2-methyl-2-pentenal, 3-octanone, ethyl acetate, 2-ethyl-furan,    2-pentyl-furan, pyrazine, 1-decanol, acetophenone, 1-nonanol,    2,5-dimethyl-pyrazine, dodecanal, benzeneacetaldehyde, nonanal,    butyrolactone, octanal, 2-decanone, hexanal, 2-nonanone,    benzaldehyde, heptanal, 2-octanone, furfural, 2-heptanone, and    pentanal.-   Embodiment 187 is the Saccharomyces cerevisiae protein composition    of any one of embodiments 170-186, wherein at least about 50% of the    protein in the protein composition falls between about 10 kDa and    about 200 kDa.-   Embodiment 188 is a Pichia pastoris protein composition comprising:    -   a plurality of functional Pichia pastoris proteins,    -   wherein the Pichia pastoris protein composition comprises at        least about 35%, on a dry weight basis, compounds larger than 5        kDa, and    -   wherein the Pichia pastoris protein composition exhibits two or        more characteristics selected from the group consisting of: 112S        is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the Pichia pastoris protein        composition, and 112S is detectable in an amount of at least        about 0.2 ppm in the headspace about 24 hours at 25° C. after        about 25 mM L-cysteine is added to the Pichia pastoris protein        composition; the Pichia pastoris protein composition forms a gel        upon heating to 65° C.; the Pichia pastoris protein composition        has a particle size distribution D10, D50, and D90 of less than        0.1 μm, 1.0 μm and 5 μm, respectively; the Pichia pastoris        protein composition is least about 80% denatured after about 20        minutes at about 85° C.; the Pichia pastoris protein composition        forms a gel with a storage modulus of at least about 100 Pa when        heated at or above about 85° C. for about 20 minutes; the Pichia        pastoris protein composition forms a gel between about pH 5.5        and about pH 10.0; the Pichia pastoris protein composition forms        a gel in solutions with ionic strength below about 0.5 M,        wherein the ionic strength is calculated based on the        concentration of non-protein solutes; and the Pichia pastoris        protein composition has an emulsion activity index of greater        than or equal to about 50 m²/g protein across about pH 4.0 to        about pH 8.0.-   Embodiment 189 is the Pichia pastoris protein composition of    embodiment 188, wherein the Pichia pastoris protein composition    exhibits three or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Pichia pastoris protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Pichia pastoris protein composition; the    Pichia pastoris protein composition forms a gel upon heating to 65°    C.; the Pichia pastoris protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Pichia pastoris protein composition is least about    80% denatured after about 20 minutes at about 85° C.; the Pichia    pastoris protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Pichia pastoris protein composition forms a gel    between about pH 5.5 and about pH 10.0; the Pichia pastoris protein    composition forms a gel in solutions with ionic strength below about    0.5 M, wherein the ionic strength is calculated based on the    concentration of non-protein solutes; and the Pichia pastoris    protein composition has an emulsion activity index of greater than    or equal to about 50 m²/g protein across about pH 4.0 to about pH    8.0.-   Embodiment 190 is the Pichia pastoris protein composition of    embodiment 188, wherein the Pichia pastoris protein composition    exhibits four or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Pichia pastoris protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Pichia pastoris protein composition; the    Pichia pastoris protein composition forms a gel upon heating to 65°    C.; the Pichia pastoris protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Pichia pastoris protein composition is least about    80% denatured after about 20 minutes at about 85° C.; the Pichia    pastoris protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Pichia pastoris protein composition forms a gel    between about pH 5.5 and about pH 10.0; the Pichia pastoris protein    composition forms a gel in solutions with ionic strength below about    0.5 M, wherein the ionic strength is calculated based on the    concentration of non-protein solutes; and the Pichia pastoris    protein composition has an emulsion activity index of greater than    or equal to about 50 m²/g protein across about pH 4.0 to about pH    8.0.-   Embodiment 191 is the Pichia pastoris protein composition of    embodiment 188, wherein the Pichia pastoris protein composition    exhibits five or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Pichia pastoris protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Pichia pastoris protein composition; the    Pichia pastoris protein composition forms a gel upon heating to 65°    C.; the Pichia pastoris protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Pichia pastoris protein composition is least about    80% denatured after about 20 minutes at about 85° C.; the Pichia    pastoris protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Pichia pastoris protein composition forms a gel    between about pH 5.5 and about pH 10.0; the Pichia pastoris protein    composition forms a gel in solutions with ionic strength below about    0.5 M, wherein the ionic strength is calculated based on the    concentration of non-protein solutes; and the Pichia pastoris    protein composition has an emulsion activity index of greater than    or equal to about 50 m²/g protein across about pH 4.0 to about pH    8.0.-   Embodiment 192 is the Pichia pastoris protein composition of    embodiment 188, wherein the Pichia pastoris protein composition    exhibits six or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Pichia pastoris protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Pichia pastoris protein composition; the    Pichia pastoris protein composition forms a gel upon heating to 65°    C.; the Pichia pastoris protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Pichia pastoris protein composition is least about    80% denatured after about 20 minutes at about 85° C.; the Pichia    pastoris protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Pichia pastoris protein composition forms a gel    between about pH 5.5 and about pH 10.0; the Pichia pastoris protein    composition forms a gel in solutions with ionic strength below about    0.5 M, wherein the ionic strength is calculated based on the    concentration of non-protein solutes; and the Pichia pastoris    protein composition has an emulsion activity index of greater than    or equal to about 50 m²/g protein across about pH 4.0 to about pH    8.0.-   Embodiment 193 is the Pichia pastoris protein composition of    embodiment 188, wherein the Pichia pastoris protein composition    exhibits seven or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Pichia pastoris protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Pichia pastoris protein composition; the    Pichia pastoris protein composition forms a gel upon heating to 65°    C.; the Pichia pastoris protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Pichia pastoris protein composition is least about    80% denatured after about 20 minutes at about 85° C.; the Pichia    pastoris protein composition forms a gel with a storage modulus of    at least about 100 Pa when heated at or above about 85° C. for about    20 minutes; the Pichia pastoris protein composition forms a gel    between about pH 5.5 and about pH 10.0; the Pichia pastoris protein    composition forms a gel in solutions with ionic strength below about    0.5 M, wherein the ionic strength is calculated based on the    concentration of non-protein solutes; and the Pichia pastoris    protein composition has an emulsion activity index of greater than    or equal to about 50 m²/g protein across about pH 4.0 to about pH    8.0.-   Embodiment 194 is the Pichia pastoris protein composition of    embodiment 188, wherein the Pichia pastoris protein composition    exhibits the characteristics:    -   H₂S is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the Pichia pastoris protein        composition, and H₂S is detectable in an amount of at least        about 0.2 ppm in the headspace about 24 hours at 25° C. after        about 25 mM L-cysteine is added to the Pichia pastoris protein        composition, wherein the Pichia pastoris protein composition        forms a gel upon heating to 65° C.,    -   wherein the Pichia pastoris protein composition has a particle        size distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm        and 5 μm, respectively,    -   wherein the Pichia pastoris protein composition is least about        80% denatured after about 20 minutes at about 85° C.,    -   wherein the Pichia pastoris protein composition forms a gel with        a storage modulus of at least about 100 Pa when heated at or        above about 85° C. for about 20 minutes,    -   wherein the Pichia pastoris protein composition forms a gel        between about pH 5.5 and about pH 10.0,    -   wherein the Pichia pastoris protein composition forms a gel in        solutions with ionic strength below about 0.5 M, wherein the        ionic strength is calculated based on the concentration of        non-protein solutes, and    -   wherein the Pichia pastoris protein composition has an emulsion        activity index of greater than or equal to about 50 m²/g protein        across about pH 4.0 to about pH 8.0.-   Embodiment 195 is the Pichia pastoris protein composition of any one    of embodiments 188-194, wherein the protein composition comprises at    least about 40%, on a dry weight basis, of compounds larger than 5    kDa.-   Embodiment 196 is the Pichia pastoris protein composition of any one    of embodiments 188-194, wherein the protein composition comprises at    least about 50%, on a dry weight basis, of compounds larger than 5    kDa.-   Embodiment 197 is the Pichia pastoris protein composition of any one    of embodiments 188-194, wherein the protein composition comprises at    least about 60%, on a dry weight basis, of compounds larger than 5    kDa.-   Embodiment 198 is the Pichia pastoris protein composition of any one    of embodiments 188-194, wherein the protein composition comprises at    least about 70%, on a dry weight basis, of compounds larger than 5    kDa.-   Embodiment 199 is the Pichia pastoris protein composition of any one    of embodiments 188-194, wherein the protein composition comprises at    least about 80%, on a dry weight basis, of compounds larger than 5    kDa.-   Embodiment 200 is the Pichia pastoris protein composition of any one    of embodiments 188-199, wherein the compounds larger than 5 kDa are    compounds larger than 10 kDa.-   Embodiment 201 is the Pichia pastoris protein composition of any one    of embodiments 188-199, wherein the compounds larger than 5 kDa are    compounds larger than 15 kDa.-   Embodiment 202 is the Pichia pastoris protein composition of any one    of embodiments 188-199, wherein the compounds larger than 5 kDa are    compounds larger than 20 kDa.-   Embodiment 203 is the Pichia pastoris protein composition of any one    of embodiments 188-199, wherein the compounds larger than 5 kDa are    compounds larger than 25 kDa.-   Embodiment 204 is the Pichia pastoris protein composition of any one    of embodiments 188-203, wherein the protein composition does not    comprise one or more compounds selected from the group consisting of    cysteine, 1-hexanol, 2-butylfuran, 2-methyl-2-pentenal, 3-octanone,    ethyl acetate, 2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol,    acetophenone, 1-nonanol, 2,5-dimethyl-pyrazine, dodecanal,    benzeneacetaldehyde, nonanal, butyrolactone, octanal, 2-decanone,    hexanal, 2-nonanone, benzaldehyde, heptanal, 2-octanone, furfural,    2-heptanone, and pentanal.-   Embodiment 205 is the Pichia pastoris protein composition of any one    of embodiments 188-204, wherein at least about 50% of the protein in    the protein composition falls between about 10 kDa and about 200    kDa.-   Embodiment 206 is an Escherichia coli protein composition    comprising:    -   a plurality of functional Escherichia coli proteins,    -   wherein the Escherichia coli protein composition comprises at        least about 35%, on a dry weight basis, compounds larger than 5        kDa, and    -   wherein the Escherichia coli protein composition exhibits two or        more characteristics selected from the group consisting of: 112S        is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the Escherichia coli protein        composition, and 112S is detectable in an amount of at least        about 0.2 ppm in the headspace about 24 hours at 25° C. after        about 25 mM L-cysteine is added to the Escherichia coli protein        composition; the Escherichia coli protein composition forms a        gel upon heating to 65° C.; the Escherichia coli protein        composition has a particle size distribution D10, D50, and D90        of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the        Escherichia coli protein composition is least about 80%        denatured after about 20 minutes at about 85° C.; the        Escherichia coli protein composition forms a gel with a storage        modulus of at least about 100 Pa when heated at or above about        85° C. for about 20 minutes; the Escherichia coli protein        composition forms a gel between about pH 5.5 and about pH 10.0;        the Escherichia coli protein composition forms a gel in        solutions with ionic strength below about 0.5 M, wherein the        ionic strength is calculated based on the concentration of        non-protein solutes; and the Escherichia coli protein        composition has an emulsion activity index of greater than or        equal to about 50 m²/g protein across about pH 4.0 to about pH        8.0.-   Embodiment 207 is the Escherichia coli protein composition of    embodiment 206, wherein the Escherichia coli protein composition    exhibits three or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Escherichia coli protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Escherichia coli protein composition; the    Escherichia coli protein composition forms a gel upon heating to 65°    C.; the Escherichia coli protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Escherichia coli protein composition is least    about 80% denatured after about 20 minutes at about 85° C.; the    Escherichia coli protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the Escherichia coli protein    composition forms a gel between about pH 5.5 and about pH 10.0; the    Escherichia coli protein composition forms a gel in solutions with    ionic strength below about 0.5 M, wherein the ionic strength is    calculated based on the concentration of non-protein solutes; and    the Escherichia coli protein composition has an emulsion activity    index of greater than or equal to about 50 m²/g protein across about    pH 4.0 to about pH 8.0.-   Embodiment 208 is the Escherichia coli protein composition of    embodiment 206, wherein the Escherichia coli protein composition    exhibits four or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Escherichia coli protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Escherichia coli protein composition; the    Escherichia con protein composition forms a gel upon heating to 65°    C.; the Escherichia coli protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Escherichia coli protein composition is least    about 80% denatured after about 20 minutes at about 85° C.; the    Escherichia coli protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the Escherichia coli protein    composition forms a gel between about pH 5.5 and about pH 10.0; the    Escherichia coli protein composition forms a gel in solutions with    ionic strength below about 0.5 M, wherein the ionic strength is    calculated based on the concentration of non-protein solutes; and    the Escherichia coli protein composition has an emulsion activity    index of greater than or equal to about 50 m²/g protein across about    pH 4.0 to about pH 8.0.-   Embodiment 209 is the Escherichia coli protein composition of    embodiment 206, wherein the Escherichia coli protein composition    exhibits five or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Escherichia coli protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Escherichia coli protein composition; the    Escherichia coli protein composition forms a gel upon heating to 65°    C.; the Escherichia coli protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Escherichia coli protein composition is least    about 80% denatured after about 20 minutes at about 85° C.; the    Escherichia coli protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the Escherichia coli protein    composition forms a gel between about pH 5.5 and about pH 10.0; the    Escherichia coli protein composition forms a gel in solutions with    ionic strength below about 0.5 M, wherein the ionic strength is    calculated based on the concentration of non-protein solutes; and    the Escherichia coli protein composition has an emulsion activity    index of greater than or equal to about 50 m²/g protein across about    pH 4.0 to about pH 8.0.-   Embodiment 210 is the Escherichia coli protein composition of    embodiment 206, wherein the Escherichia coli protein composition    exhibits six or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Escherichia coli protein    composition, and 112S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Escherichia coli protein composition; the    Escherichia coli protein composition forms a gel upon heating to 65°    C.; the Escherichia coli protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Escherichia coli protein composition is least    about 80% denatured after about 20 minutes at about 85° C.; the    Escherichia coli protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the Escherichia coli protein    composition forms a gel between about pH 5.5 and about pH 10.0; the    Escherichia coli protein composition forms a gel in solutions with    ionic strength below about 0.5 M, wherein the ionic strength is    calculated based on the concentration of non-protein solutes; and    the Escherichia coli protein composition has an emulsion activity    index of greater than or equal to about 50 m²/g protein across about    pH 4.0 to about pH 8.0.-   Embodiment 211 is the Escherichia coli protein composition of    embodiment 206, wherein the Escherichia coli protein composition    exhibits seven or more characteristics selected from the group    consisting of: H₂S is detectable in an amount of less than about 0.1    ppm when L-cysteine is not added to the Escherichia coli protein    composition, and H₂S is detectable in an amount of at least about    0.2 ppm in the headspace about 24 hours at 25° C. after about 25 mM    L-cysteine is added to the Escherichia coli protein composition; the    Escherichia coli protein composition forms a gel upon heating to 65°    C.; the Escherichia coli protein composition has a particle size    distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm,    respectively; the Escherichia coli protein composition is least    about 80% denatured after about 20 minutes at about 85° C.; the    Escherichia coli protein composition forms a gel with a storage    modulus of at least about 100 Pa when heated at or above about    85° C. for about 20 minutes; the Escherichia coli protein    composition forms a gel between about pH 5.5 and about pH 10.0; the    Escherichia coli protein composition forms a gel in solutions with    ionic strength below about 0.5 M, wherein the ionic strength is    calculated based on the concentration of non-protein solutes; and    the Escherichia coli protein composition has an emulsion activity    index of greater than or equal to about 50 m²/g protein across about    pH 4.0 to about pH 8.0.-   Embodiment 212 is the Escherichia coli protein composition of    embodiment 206, wherein the Escherichia coli protein composition    exhibits the characteristics:    -   H₂S is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the Escherichia coli protein        composition, and H₂S is detectable in an amount of at least        about 0.2 ppm in the headspace about 24 hours at 25° C. after        about 25 mM L-cysteine is added to the Escherichia coli protein        composition,    -   wherein the Escherichia coli protein composition forms a gel        upon heating to 65° C.,    -   wherein the Escherichia coli protein composition has a particle        size distribution D10, D50, and D90 of less than 0.1 μm, 1.0 μm        and 5 μm, respectively,    -   wherein the Escherichia coli protein composition is least about        80% denatured after about 20 minutes at about 85° C.,    -   wherein the Escherichia coli protein composition forms a gel        with a storage modulus of at least about 100 Pa when heated at        or above about 85° C. for about 20 minutes,    -   wherein the Escherichia coli protein composition forms a gel        between about pH 5.5 and about pH 10.0,    -   wherein the Escherichia coli protein composition forms a gel in        solutions with ionic strength below about 0.5 M, wherein the        ionic strength is calculated based on the concentration of        non-protein solutes, and    -   wherein the Escherichia coli protein composition has an emulsion        activity index of greater than or equal to about 50 m²/g protein        across about pH 4.0 to about pH 8.0.-   Embodiment 213 is the Escherichia coli protein composition of any    one of embodiments 206-212, wherein the protein composition    comprises at least about 40%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 214 is the Escherichia coli protein composition of any    one of embodiments 206-212, wherein the protein composition    comprises at least about 50%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 215 is the Escherichia coli protein composition of any    one of embodiments 206-212, wherein the protein composition    comprises at least about 60%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 216 is the Escherichia coli protein composition of any    one of embodiments 206-212, wherein the protein composition    comprises at least about 70%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 217 is the Escherichia coli protein composition of any    one of embodiments 206-216, wherein the protein composition    comprises at least about 80%, on a dry weight basis, of compounds    larger than 5 kDa.-   Embodiment 218 is the Escherichia coli protein composition of any    one of embodiments 206-216, wherein the compounds larger than 5 kDa    are compounds larger than 10 kDa.-   Embodiment 219 is the Escherichia coli protein composition of any    one of embodiments 206-216, wherein the compounds larger than 5 kDa    are compounds larger than 15 kDa.-   Embodiment 220 is the Escherichia coli protein composition of any    one of embodiments 206-216, wherein the compounds larger than 5 kDa    are compounds larger than 20 kDa.-   Embodiment 221 is the Escherichia coli protein composition of any    one of embodiments 206-216, wherein the compounds larger than 5 kDa    are compounds larger than 25 kDa.-   Embodiment 222 is the Escherichia coli protein composition of any    one of embodiments 206-221, wherein the protein composition does not    comprise one or more compounds selected from the group consisting of    cysteine, 1-hexanol, 2-butylfuran, 2-methyl-2-pentenal, 3-octanone,    ethyl acetate, 2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol,    acetophenone, 1-nonanol, 2,5-dimethyl-pyrazine, dodecanal,    benzeneacetaldehyde, nonanal, butyrolactone, octanal, 2-decanone,    hexanal, 2-nonanone, benzaldehyde, heptanal, 2-octanone, furfural,    2-heptanone, and pentanal.-   Embodiment 223 is the Escherichia coli protein composition of any    one of embodiments 206-222, wherein at least about 50% of the    protein in the protein composition falls between about 10 kDa and    about 200 kDa.-   Embodiment 224 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate to obtain a filtered lysate;    -   d) concentrating the filtered lysate to obtain a protein        composition; and    -   e) optionally pasteurizing the protein composition of protein to        obtain a pasteurized protein composition,    -   wherein steps a), b), c), d), and e) independently, are        performed at a pH between about 8.5 and about 12.0.-   Embodiment 225 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) concentrating the clarified lysate to obtain a protein        composition; and    -   d) optionally pasteurizing the protein composition of protein to        obtain a pasteurized protein composition,    -   wherein steps a), b), c), and d) independently, are performed at        a pH between about 8.5 and about 12.0.-   Embodiment 226 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate to obtain a protein        composition; and    -   d) optionally pasteurizing the protein composition, to obtain a        pasteurized protein composition,    -   wherein steps a), b), c), and d) independently, are performed at        a pH between about 8.5 and about 12.0.-   Embodiment 227 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate using microfiltration to        obtain a first filtered lysate;    -   d) filtering the first filtered lysate using diafiltration to        obtain a second filtered lysate;    -   e) filtering the second filtered lysate using ultrafiltration        obtain a third filtered lysate;    -   f) filtering the third filtered lysate using diafiltration to        obtain a protein composition; and    -   g) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), c), d), e), f), and g) independently, are        performed at a pH between about 8.5 and about 12.0.-   Embodiment 228 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) clarifying the cell lysate, optionally in the presence of one        or more flocculants, to obtain a clarified lysate;    -   c) filtering the clarified lysate using ultrafiltration to        obtain a first filtered lysate;    -   d) filtering the first filtered lysate using diafiltration to        obtain a protein composition; and    -   e) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), c), d), and e) independently, are        performed at a pH between about 8.5 and about 12.0.-   Embodiment 229 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) filtering the cell lysate to obtain a protein composition;        and    -   c) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), and c) independently, are performed at a        pH between about 8.5 and about 12.0.-   Embodiment 230 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) filtering the cell lysate using ultrafiltration obtain a        first filtered lysate;    -   c) filtering the first filtered lysate using diafiltration to        obtain a protein composition; and    -   d) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a) b), c), and d) independently, are performed at        a pH between about 8.5 and about 12.0.-   Embodiment 231 is a protein composition produced by a method    comprising:    -   a) lysing an aqueous suspension of the plurality of cells to        obtain a cell lysate;    -   b) filtering the cell lysate using ultrafiltration to obtain a        protein composition; and    -   c) optionally pasteurizing the protein composition to obtain a        pasteurized protein composition,    -   wherein steps a), b), and c) independently, are performed at a        pH between about 8.5 and about 12.0.-   Embodiment 232 is the protein composition of any one of embodiments    224-228 wherein the clarifying step is performed, optionally in the    presence of one or more flocculants, at a pH between about 9.0 and    about 12.0.-   Embodiment 233 is the protein composition of embodiment 232, wherein    the clarifying step is performed at a pH between about 9.0 and about    10.0.-   Embodiment 234 is the protein composition of embodiment 232, wherein    the clarifying step is performed at a pH between about 10.0 and    about 11.0.-   Embodiment 235 is the protein composition of embodiment 232, wherein    the clarifying step is performed at a pH between about 11.0 and    about 12.0.-   Embodiment 236 is the protein composition of any one of embodiments    224-228 or 232-235, wherein clarifying step is performed by    centrifugation to less than about 20% dry solids.-   Embodiment 237 is the protein composition of any one of embodiments    224-228 or 232-236, wherein the clarifying step is performed by    gravity settling to less than about 20% dry solids.-   Embodiment 238 is the protein composition of any one of embodiments    224-228 or 232-237, wherein the clarifying step is performed by    diatomaceous earth filtration to less than about 20% dry solids.-   Embodiment 239 is the protein composition of any one of embodiments    224-228 or 232-238, wherein the lysate is diluted 1:1 with water or    aqueous solution of salt or buffer before clarifying, wherein the pH    is between about 8.5 and about 12.0.-   Embodiment 240 is the protein composition of any one of embodiments    224-228 or 232-239, wherein the cell lysate from step a) is    clarified in the presence of one or more flocculants.-   Embodiment 241 is the protein composition of embodiment 240, wherein    the one or more flocculants comprise one or more of alkylamine    epichlorohydrin, polydimethyldiallylammonium chloride, a polyamine,    lime, hydrated lime, ferric chloride, ferric sulfate, ferrous    sulfate, aluminum sulfate, sodium aluminate, aluminum chloride,    magnesium carbonate hydroxide, calcium carbonate, calcium hydroxide,    an activated silicate, a guar gum, a starch, a tannin, sodium    alginate, polyaluminum sulfate, polyaluminum hydroxy chloride,    BIO-FLOCK®, and a synthetic polyelectrolyte.-   Embodiment 242 is the protein composition of embodiment 240, wherein    the one or more flocculants are selected from the group consisting    of alkylamine epichlorohydrin, polydimethyldiallylammonium chloride,    a polyamine, lime, hydrated lime, ferric chloride, ferric sulfate,    ferrous sulfate, aluminum sulfate, sodium aluminate, aluminum    chloride, magnesium carbonate hydroxide, calcium carbonate, calcium    hydroxide, an activated silicate, a guar gum, a starch, a tannin,    sodium alginate, polyaluminum sulfate, polyaluminum hydroxy    chloride, BIO-FLOCK®, and a synthetic polyelectrolyte.-   Embodiment 243 is the method of any one of embodiments 224-242,    wherein filtering comprises microfiltration.-   Embodiment 244 is the method of any one of embodiments 224-243,    wherein filtering comprises ultrafiltration.-   Embodiment 245 is the method of any one of embodiments 224-244,    wherein filtering comprises diafiltration.-   Embodiment 246 is the method of embodiment 245, where diafiltration    is performed for at least two diavolumes.-   Embodiment 247 is the protein composition of any one of embodiments    224-246, wherein the plurality of cells comprises microbial cells.-   Embodiment 248 is the protein composition of any one of embodiments    224-247, wherein the plurality of cells comprises fungal cells.-   Embodiment 249 is the protein composition of embodiment 248, wherein    the fungal cells are selected from the group consisting of    Saccharomyces, Pichia, Candida, Hansenula, Torulopsis,    Kluyveromyces, Yarrowia, and Fusarium cells.-   Embodiment 250 is the protein composition of embodiment 248, wherein    the fungal cells are selected from the group consisting of    Saccharomyces cerevisiae, Pichia pastoris, Candida boidinii,    Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, and    Fusarium venenatum.-   Embodiment 251 is the protein composition of any one of embodiments    224-250, wherein the plurality of cells comprises bacterial cells.-   Embodiment 252 is the protein composition of embodiment 251, wherein    the bacterial cells are selected from the group consisting of    Bacillus, Escherichia, Lactobacillus, Corynebacterium, Pseudomonas,    and Methanococcus.-   Embodiment 253 is the protein composition of embodiment 251, wherein    the bacterial cells are selected from the group consisting of    Escherichia coli, Bacillus subtilis, Lactobacillus lactis,    Corynebacterium glutamicum, Pseudomonas fluorescens, and    Methanococcus maripaludis.-   Embodiment 254 is the protein composition of any one of embodiments    224-253, wherein the aqueous suspension of the plurality of cells    comprises from about 2% to about 25% dry solids.-   Embodiment 255 is the protein composition of any one of embodiments    224-254, further comprising washing the aqueous suspension of the    plurality of cells at a pH between about 8.5 and about 12.0 before    step a).-   Embodiment 256 is the protein composition of any one of embodiments    224-255, wherein the lysing step is performed at a temperature    between about 4° C. and about 15° C.-   Embodiment 257 is the protein composition of any one of embodiments    224-256, wherein the lysing step is performed biochemically.-   Embodiment 258 is the protein composition of any one of embodiments    224-257, wherein the lysing step is performed chemically.-   Embodiment 259 is the protein composition of any one of embodiments    224-258, wherein the lysing step is performed mechanically.-   Embodiment 260 is the protein composition of any one of embodiments    224-259, wherein the lysing step is performed at a pH between about    9.0 and about 12.0.-   Embodiment 261 is the protein composition of embodiment 260, wherein    the lysing step is performed at a pH between about 9.0 and about    10.0.-   Embodiment 262 is the protein composition of embodiment 260, wherein    the lysing step is performed at a pH between about 10.0 and about    11.0.-   Embodiment 263 is the protein composition of embodiment 260, wherein    the lysing step is performed at a pH between about 11.0 and about    12.0.-   Embodiment 264 is the protein composition of any one of embodiments    224-263, wherein the lysate is diluted 1:1 with water or aqueous    solution of salt or buffer before filtering, wherein the pH is    between about 8.5 and about 12.0.-   Embodiment 265 is the protein composition of any one of embodiments    224-264, wherein the protein composition has a protein content of    about 2 mg/mL to about 250 mg/mL.-   Embodiment 266 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits one or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 267 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits two or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 268 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits three or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 269 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits four or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 270 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits five or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 271 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits six or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 272 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits seven or more    characteristics selected from the group consisting of: H₂S is    detectable in an amount of less than about 0.1 ppm when L-cysteine    is not added to the protein composition, and H₂S is detectable in an    amount of at least about 0.2 ppm in the headspace about 24 hours at    25° C. after about 25 mM L-cysteine is added to the protein    composition; the protein composition forms a gel upon heating to 65°    C.; the protein composition has a particle size distribution D10,    D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively; the    protein composition is least about 80% denatured after about 20    minutes at about 85° C.; the protein composition forms a gel with a    storage modulus of at least about 100 Pa when heated at or above    about 85° C. for about 20 minutes; the protein composition forms a    gel between about pH 5.5 and about pH 10.0; the protein composition    forms a gel in solutions with ionic strength below about 0.5 M,    wherein the ionic strength is calculated based on the concentration    of non-protein solutes; and the protein composition has an emulsion    activity index of greater than or equal to about 50 m²/g protein    across about pH 4.0 to about pH 8.0.-   Embodiment 273 is the protein composition of any one of embodiments    224-265, wherein the protein composition exhibits the    characteristics:    -   H₂S is detectable in an amount of less than about 0.1 ppm when        L-cysteine is not added to the protein composition, and H₂S is        detectable in an amount of at least about 0.2 ppm in the        headspace about 24 hours at 25° C. after about 25 mM L-cysteine        is added to the protein composition,    -   the protein composition forms a gel upon heating to 65° C.,    -   the protein composition has a particle size distribution D10,        D50, and D90 of less than 0.1 μm, 1.0 μm and 5 μm, respectively,    -   the protein composition is least about 80% denatured after about        20 minutes at about 85° C.,    -   the protein composition forms a gel with a storage modulus of at        least about 100 Pa when heated at or above about 85° C. for        about 20 minutes,    -   wherein the protein composition forms a gel between about pH 5.5        and about pH 10.0,    -   the protein composition forms a gel in solutions with ionic        strength below about 0.5 M, wherein the ionic strength is        calculated based on the concentration of non-protein solutes,        and    -   the protein composition has an emulsion activity index of        greater than or equal to about 50 m²/g protein across about pH        4.0 to about pH 8.0.-   Embodiment 274 is the protein composition of any one of embodiments    224-273, wherein the protein composition comprises at least about    35%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 275 is the protein composition of any one of embodiments    224-273, wherein the protein composition comprises at least about    40%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 276 is the protein composition of any one of embodiments    224-273, wherein the protein composition comprises at least about    50%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 277 is the protein composition of any one of embodiments    224-273, wherein the protein composition comprises at least about    60%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 278 is the protein composition of any one of embodiments    224-273, wherein the protein composition comprises at least about    70%, on a dry weight basis, of compounds larger than 5 kDa.-   Embodiment 279 is the protein composition of any one of embodiments    274-278, wherein the compounds larger than 5 kDa are compounds    larger than 10 kDa.-   Embodiment 280 is the protein composition of any one of embodiments    274-278, wherein the compounds larger than 5 kDa are compounds    larger than 15 kDa.-   Embodiment 281 is the protein composition of any one of embodiments    274-278, wherein the compounds larger than 5 kDa are compounds    larger than 20 kDa.-   Embodiment 282 is the protein composition of any one of embodiments    274-278, wherein the compounds larger than 5 kDa are compounds    larger than 25 kDa.-   Embodiment 283 is the protein composition of any one of embodiments    224-282, further comprising drying the protein composition.-   Embodiment 284 is the protein composition of embodiment 283, wherein    the protein composition is spray dried.-   Embodiment 285 is the protein composition of embodiment 283, wherein    the protein composition is freeze dried.-   Embodiment 286 is the protein composition of any one of embodiments    224-282, further comprising pasteurizing the protein composition to    obtain a pasteurized protein composition.-   Embodiment 287 is the protein composition of embodiment 286, wherein    the protein composition is pasteurized by microfiltration.-   Embodiment 288 is the protein composition of embodiment 286, wherein    protein composition is pasteurized by high temperature short time    pasteurization.-   Embodiment 289 is the protein composition of embodiment 286, wherein    the protein composition is pasteurized by adding one or more    antimicrobials.-   Embodiment 290 is the protein composition of any one of embodiments    286-289, further comprising drying the pasteurized protein    composition.-   Embodiment 291 is the protein composition of embodiment 290, wherein    the pasteurized protein composition is spray dried.-   Embodiment 292 is the protein composition of embodiment 290, wherein    the pasteurized protein composition is freeze dried.-   Embodiment 293 is the protein composition of any one of embodiments    224-292, wherein the amount of one or more volatile compounds is    reduced by at least about 1.05-fold compared to a corresponding    method in which one or more of the lysing, clarifying, or filtering    steps are not performed at a pH between about 8.5 and about 12.0,    wherein the volatile compound is selected from the group consisting    of cysteine, 1-hexanol, 2-butylfuran, 2-methyl-2-pentenal,    3-octanone, ethyl acetate, 2-ethyl-furan, 2-pentyl-furan, pyrazine,    1-decanol, acetophenone, 1-nonanol, 2,5-dimethyl-pyrazine,    dodecanal, benzeneacetaldehyde, nonanal, butyrolactone, octanal,    2-decanone, hexanal, 2-nonanone, benzaldehyde, heptanal, 2-octanone,    furfural, 2-heptanone, and pentanal.-   Embodiment 294 is the protein composition of any one of embodiments    224-293, wherein the protein composition does not comprise one or    more compounds selected from the group consisting of cysteine,    1-hexanol, 2-butylfuran, 2-methyl-2-pentenal, 3-octanone, ethyl    acetate, 2-ethyl-furan, 2-pentyl-furan, pyrazine, 1-decanol,    acetophenone, 1-nonanol, 2,5-dimethyl-pyrazine, dodecanal,    benzeneacetaldehyde, nonanal, butyrolactone, octanal, 2-decanone,    hexanal, 2-nonanone, benzaldehyde, heptanal, 2-octanone, furfural,    2-heptanone, and pentanal.-   Embodiment 295 is the protein composition of any one of embodiments    224-294, wherein at least about 50% of the protein in the protein    composition falls between about 10 kDa and about 200 kDa-   Embodiment 296 is a food product comprising:    -   a protein composition of any one of embodiments 129-169.-   Embodiment 297 is a food product comprising:    -   a Saccharomyces cerevisiae protein composition of any one of        embodiments 170-187.-   Embodiment 298 is a food product comprising:    -   a Pichia pastoris protein composition of any one of embodiments        188-205.-   Embodiment 299 is a food product comprising:    -   an Escherichia coli protein composition of any one of        embodiments 206-223.-   Embodiment 300 is the food product of any one of embodiments    296-290, wherein the food product is a dairy replica.-   Embodiment 301 is the food product of embodiment 300, wherein the    food product is a milk replica.-   Embodiment 302 is the food product of embodiment 300, wherein the    food product is a cheese replica.-   Embodiment 303 is the food product of any one of embodiments    300-302, wherein the food product further comprises one or more    microbes.-   Embodiment 304 is the food product of embodiment 303, wherein the    one or more microbes are selected from the group consisting of a    Lactococcus species, a Lactobacillus species, a Leuconostocaceae    species, a Streptococcus species, a Pediococcus species, a    Clostridium species, a Staphylococcus species, a Brevibacterium    species, a Propioniibacteria species, a Penicillium species, a    Debaryomyces, a Geotrichum species, a Corynebacteria species, a    Verticillium species, a Kluyveromyces species, a Saccharomyces    species, a Candida species, a Rhodosporidum species, a Micrococcus    species, a Halomonas species, a Psychrobacter species, or a    combination thereof.-   Embodiment 305 is the food product of embodiment 303, wherein the    one or more microbes are selected from the group consisting of    Lactococcus lactis lactis, Lactococcus lactis cremoris, Lactococcus    lactis biovar diacetylactis, Lactobacillus delbrueckii lactis,    Lactobacillus delbrueckii bulgaricus, Lactobacillus helveticus,    Lactobacillus plantarum, Lactobacillus casei, Lactobacillus    rhamnosus, Leuconostoc mesenteroides cremoris, Streptococcus    thermophiles, Pediococcus pentosaceus, Clostridium butyricum,    Staphylococcus xylosus, Brevibacterium linens, Penicillium candidum,    Penicillium camemberti, Penicillium roqueforti, Debaryomyces    hansenii, Geotrichum candidum, Verticillium lecanii, Kluyveromyces    lactis, Saccharomyces cerevisiae, Candida jefer, Candida utilis,    Rhodosporidum infirmominiatum,-   Embodiment 306 is the food product of any one of embodiments    296-299, wherein the food product is a meat replica.-   Embodiment 307 is the food product of embodiment 306, further    comprising a heme.-   Embodiment 308 is the food product of embodiment 308, wherein the    heme is provided in the form of a heme-containing protein.-   Embodiment 309 is the food product of any one of embodiments    306-308, further comprising one or more flavor precursors.-   Embodiment 310 is the food product of embodiment 309 wherein the one    or more flavor precursors comprise a compound selected from the    group consisting of a sugar, a sugar alcohol, a sugar acid, a sugar    derivative, a sulfur-containing compound, an amino acids or    derivative thereof, a nucleotide, a nucleoside, a vitamin, an acid,    a peptides, a protein hydrolysate, an extract, and combinations    thereof.-   Embodiment 311 is the food product of embodiment 309, wherein the    flavor precursors comprise a sugar and a sulfur-containing compound.-   Embodiment 312 is the food product of any one of embodiments    310-311, wherein the sulfur-containing compound is selected from the    group consisting of cysteine, cystine, a cysteine sulfoxide,    allicin, selenocysteine, methionine, thiamine, and combinations    thereof.-   Embodiment 313 is the food product of any one of embodiments    310-312, wherein the sugar is selected from the group consisting of    glucose, fructose, ribose, sucrose, arabinose, glucose-6-phosphate,    fructose-6-phosphate, fructose 1,6-diphosphate, inositol, maltose,    molasses, maltodextrin, glycogen, galactose, lactose, ribitol,    gluconic acid and glucuronic acid, amylose, amylopectin, xylose, and    combinations thereof.-   Embodiment 314 is the food product of any one of embodiments    306-313, further comprising an oil.-   Embodiment 315 is the food product of embodiment 314, wherein the    oil is selected from the group consisting of coconut oil, mango oil,    sunflower oil, cottonseed oil, safflower oil, rice bran oil, cocoa    butter, palm fruit oil, palm oil, soybean oil, canola oil, corn oil,    sesame oil, walnut oil, flaxseed, jojoba oil, castor, grapeseed oil,    peanut oil, olive oil, algal oil, oil from bacteria or fungi, and    combinations thereof.-   Embodiment 316 is the food product of any one of embodiments    296-299, wherein the food product is a protein supplement.-   Embodiment 317 is the food product of any one of embodiments    296-316, wherein the food product contains less than 10% (by weight    of the food product) animal products.-   Embodiment 318 is the food product of any one of embodiments    296-316, wherein the food product contains less than 5% (by weight    of the food product) animal products.-   Embodiment 319 is the food product of any one of embodiments    296-316, wherein the food product contains less than 1% (by weight    of the food product) animal products.-   Embodiment 320 is the food product of any one of embodiments    296-316, wherein the food product contains no animal products.-   Embodiment 321 is the food product of any one of embodiments    296-316, wherein the food product contains less than 10% (by weight    of the food product) animal-derived products.-   Embodiment 322 is the food product of any one of embodiments    296-316, wherein the food product contains less than 5% (by weight    of the food product) animal-derived products.-   Embodiment 323 is the food product of any one of embodiments    296-316, wherein the food product contains less than 1% (by weight    of the food product) animal-derived products.-   Embodiment 324 is the food product of any one of embodiments    296-316, wherein the food product contains no animal-derived    products.-   Embodiment 325 is the food product of any one of embodiments    296-316, wherein the food product contains less than 10% (by weight    of the food product) animal meat.-   Embodiment 326 is the food product of any one of embodiments    296-316, wherein the food product contains less than 5% (by weight    of the food product) animal meat.-   Embodiment 327 is the food product of any one of embodiments    296-316, wherein the food product contains less than 1% (by weight    of the food product) animal meat.-   Embodiment 328 is the food product of any one of embodiments    296-316, wherein the food product contains no animal meat.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Lysis of Yeast Cells at pH 6.0 or 9.0

Aqueous suspensions of S. cerevisiae were prepared using yeast crumble(Cool Smart Yeast, LeSaffre Yeast Corporation Item No #05518) at 1:1crumble:MILLI-Q® water at either pH 6.0 or pH 9.0. The yeast crumbleswere lysed using bead milling while maintaining a pH or either 6.0 or9.0, as appropriate. The lysed cells were clarified by centrifuging at8,000×g for 3 minutes using a small-scale model of a pilot scaledisc-stack centrifuge. The clarified lysate was filtered.

The use of pH 9.0 versus 6.0 had several desirable results. Aftercentrifugation, the clarified lysate maintained at pH 9.0 had a 25-50%increase in yield relative to the lysate maintained at pH 6.0 (e.g., inone experiment, pH 6.0 gave 10% dry-basis (DB) yield vs. 15% DB yield atpH 9.0) and a 30% decrease in protein loss during microfiltration (0.2μm membrane). At the sensory level, the development of undesirableoff-flavors in supernatant/centrate were significantly reduced, suchthat a panel of experienced sniffers were immediately able to sortblinded samples.

Example 2 Lysis of Yeast Cells at pH 6.0 or 9.0

Aqueous suspensions of P. pastoris expressing soybean leghemoglobin wereprepared using broth fermented by Impossible Foods. Whole cells wereisolated by centrifugation using a pilot-scale disk stack centrifuge(Alfa Laval BRPX 810 SGV-34CG; feed rate: 10 LPM; initial dischargetimer: 5 min; feed % SS: 10-13; centrate solids % SS: 55-60; 2-10° C.).Cells were resuspended at 1:1 ratio using deionized water (2-10° C.).Cell suspension pH was adjusted to pH 6.0 or 9.0 using 5M HCl or 5M NaOHuntil cell suspension pH was stable for 30 minutes. Cell suspensionswere lysed using a small-scale homogenizer (Gaulin 30CD; 13,000-15,000psi; 3 passes) with cooling to 2-10° C. and pH adjustment betweenpasses. The lysed cells were clarified by centrifuging at 12,000×g for20 minutes using a small-scale model of a pilot scale disc-stackcentrifuge. Clarified lysate (“centrate”) was applied directly to ahollow-fiber ultrafiltration membrane (Koch Romicon part #0721039; 30kDa molecular weight cutoff (MWCO), 1.1 mm diameter fibers),concentrated to −10% dry solids (DS) and diafiltered using deionizedwater at either pH 6.0 or pH 9.0. Final product was obtained afterdrying in a lyophilizer. After drying, 10% (w/v) suspensions using eachfinal product were prepared at a final pH of approximately 7.0 andanalyzed using a hybrid rheometer (TA Instruments, DHR series; 4C/minsteps).

The use of pH 9.0 versus 6.0 had several desirable results. Aftercentrifugation, the clarified lysate maintained at pH 9.0 had a 35%increase in protein yield relative to the lysate maintained at pH 6.0.When heated to 95° C. at 10% (w/v) DS, thermally-set gels using the pH9.0 final product were about 10-fold stronger (i.e., higher storagemodulus) than those obtained from final product from the pH 6.0 process.

Example 3 Lysis of Bacterial Cells at pH 6.0 or 9.0

Cultures of E. coli cells (DH5alpha, 8 liters total) were prepared bygrowth in lysogeny broth (LSB media) at 37° C. in shake flasks. Wholecells were isolated by centrifugation at 15,000×g for 20 minutes using afloor model laboratory centrifuge.

Cells were resuspended at 1:5 ratio (grams cell pellet:mL water) usingMILLI-Q® water (2-10° C.). Cell suspension was split into two halves,with pH was adjusted to pH 6.0 or 9.0 using 5M HCl or 5M NaOH until cellsuspension pH was stable for 30 minutes. Cell suspensions were lysedusing a small-scale homogenizer (Gaulin 30CD; 13,000-15,000 psi; 3passes) with cooling to 2-10° C. and pH adjustment between passes. Thelysed cells were dialyzed directly on 30 kDa dialysis membranes (PierceSlide-a-Lyzer)

The use of pH 9.0 versus 6.0 had several desirable results: 1) proteinreleased after cell lysis increased by 30%; 2) storage modulus(firmness) increase of thermally-set gels improved by about 6-fold(final product of Process Variant C, pH 9 vs. pH 6; 10% w/v suspensionsprepared in MILLI-Q® water to pH 7.0; assayed by rheometer as describedpreviously); key odor-active volatiles from a range of classes weredecreased by 30-75% (e.g., 3-octanone, ethyl acetate, pyrazine, nonanal,acetaldehyde).

Example 4 Purification of Protein at pH 6.5 or 9.5

Total cellular protein was purified from cultures of S. cerevisiae intwo different experiments using the same methods and materials, exceptthe pH of the solutions throughout the purification were at either pH6.5 or pH 9.5. Aqueous suspensions of S. cerevisiae were prepared usingyeast crumble (Cool Smart Yeast, LeSaffre Yeast Corporation Item No#05518) at 1:1 crumble:MILLI-Q® water. The yeast crumbles were lysedusing bead milling while maintaining a pH of or either 6.5 or 9.5, asappropriate, to make cell lysates. The lysates were clarified bycentrifuging at 8,000×g for 3 minutes using a small-scale model of apilot scale disc-stack centrifuge. The clarified lysates (centrates)were incubated at 4° C. overnight. A panel of trained tasters andsmellers then tested the resulting product after cold storage. The panelwas able to correctly sort the pH 6.5 from the pH 9.5 process samples byscoring off-odors in the absence of other visual cues.

Example 5 Filtration, Concentration and/or Desalting of ProteinConcentration or Isolate at pH 9.5

Lysis of yeast cells was performed as indicated in previous examples.The material may be optionally microfiltered to remove in-processmicrobial counts (e.g., using a WaterSep Mini-BioProducer41, HF 0.2 μmmicrofilter, cat WA 920 10MPR41 SG), yielding the improved proteinpassage of 30%. With or without microfiltration, the material may beconcentrated, diafiltered and depleted of small molecules usingultrafiltration. For example, the material has been applied to bothWaterSep (cat: BC 030 20GRA43 1L) and Koch (cat: HF, 6043-97-43-Pf030)using 4-10 diavolumes prior to a final concentration down to 10-16% drysolids. The alkaline processing method described produced a superioringredient that demonstrates improved food activities: firmer gels uponcooking (FIGS. 3-5 ) with lower off-odor and off-flavor (See, e.g.,Table 4). Table 2 lists some exemplary specifications of proteinproduced by this process. Table 3 lists some exemplary benefits of thepH 9.5 technique compared to the pH 6.5 technique. Table 4 lists someexemplary compounds depleted by the pH 9.5 technique.

TABLE 2 Material Specifications Property Assay Specification ProteinDifferential Scanning >80% detectable Denaturation Fluorimetryhydrophobic exposure is complete between 50° C. and 85° C.; maximalhydrophobic exposure occurs between 50° C. and 75° C.; pH 5.5-10.0;non-protein ionic strength 0-0.5 M Gelation Hybrid Rheometer 10% (w/v)suspension gels to 100 Pa storage modulus when heated to 95° C. andcooled back to 25° C. Particle Size Laser diffraction D10 < 0.1 μm;Distribution (Mastersizer, Malvern) D50 < 1.0 μm; D90 < 5 μm PolypeptideReducing, denaturing Greater than 50% of Integrity SDS-PAGECoommassie-stained polypeptides fall between 10 kDa and 200 kDa asmeasured by densitometry H₂S release Hach Hydrogen No added cysteine:Sulfide Test Kit H₂S < 0.1 ppm (Cat. No. 25379-00). 25 mM addedcysteine: Test 45 mL head space H₂S > 0.2 ppm in 50 ml Falcon tube with5 mL product, 2% (w/v, aqueous), pH 7.0 at RT × 24 hours. Filters fittedunder cap. No effervescent tablet added Buffer Buffer capacity less than2.5 mmol Capacity measured by titration NaOH per with NaOH or HCl gramdry solids between pH 3 and pH 12 Volatile GCMS on pH- and >2.5 Xreduction Aroma volume-matched sample in the following Compoundsvolatile compounds: 3-Octanone; Acetophenone; 1-Nonanol;Benzeneacetaldehyde; Nonanal, when headspace of a 2 mL of a 10% (w/v)suspension is assayed in a 20 mL glass vial at 50 C. by adsorption ontoSPME fiber, followed by GCMS Emulsion Mechanical >50 m²/g proteinActivity homogenization across pH 4.0-8.0 Index of aqueous proteinsolution with canola oil; EAI = 2 T)/ (c*vol_fx_oil) where T =ln(10)*A₆₀₀/path_length

TABLE 3 Process and composition benefits obtained using alkaline processtechnique combined with filtration Process or composition featureBenefit (pH 9.5 vs. pH 6.5) Protein Released 25-50% increaseMicrofiltration performance 30% increase in permeated protein Solidsremoval (e.g., by >20% recovery of aqueous phase volume centrifugation)Gel strength at 10% (w/v) ≥10-fold increase in storage modulus at 95° C.Buffer capacity of Less than or equal to the equivalent of 2.5isolate/concentrate mmol NaOH per gram dry solids required to shift pHfrom pH 3.0-pH 12.0 of material at 2% dry solids (w/v) in MILLI-Q ®water. Gelation capacity after ≥2-fold increase in storage modulus atpasteurization; 95° C. PZ at 65° C. × 30 sec assayed at 10% (w/v) drysolids

TABLE 4 Small Molecules depleted using alkaline processing techniquePichia Pichia Saccharomyces Saccharomyces processed processed processedprocessed without with Pichia without with Saccharomyces alkalinealkaline Factor alkaline alkaline factor Odor Compound process processChange process process change Descriptor 3-Octanone 1,661,170.00 169,8819.78 Not Detected Not Detected Not Detected Musty, mushroom, ketonic,moldy and cheesy fermented with a green, vegetative nuance Ethyl Acetate15,927,040.00 5,408,039 2.95 Not Detected Not Detected Not DetectedEtherial, fruity, sweet, with a grape and cherry nuance Pyrazine2,592,053 1,656,755 1.56 Not Detected Not Detected Not Detected pungentsweet corn like roasted hazelnut barly Acetophenone 9,416,779 321,95329.25 120,697,700 9,104,363 13.25 Powdery, bitter almond cherry pit-like with coumarinic and fruity nuances 1-Nonanol 831,649 Not DetectedINF 92,858,790 9,737,588 9.53 fresh clean fatty floral rose orange dustywet oily 2,5-dimethyl- Not Detected Not Detected Not Detected 3,615,044405,892 8.90 Nutty, Pyrazine peanut, musty, earthy, powdery and slightlyroasted with a cocoa powder nuance Nonanal 2,819,845 456,452 6.18467,067,300 186,830,400 2.49 waxy aldehydic rose fresh orris orange peelfatty peely 2-Decanone 592,550 Not Detected INF 2,333,218 2,163,738 1.07orange floral fatty peach Listed values correspond to peak integrationareas as described in the data processing in example 4. Factor change =(compound area of non alkaline process/compound area of alkalineprocess) INF = factor change unable to be determined

Example 6 Measurement of Polypeptide Integrity in Protein Compositions

Dried protein compositions from using Process Variants B or C (FIG. 2 ),from S. cerevisiae, P. pastoris or E. coli, were brought to 10% (w/v)final suspension of material in MILLI-Q® water. The pH was adjusted topH 9.0 using NaOH or HCl and protein concentration was measured usingthe Pierce 660 nm Protein Assay Reagent (cat #22660), following themanufacturer's instructions. Suspensions were then adjusted to finalprotein concentration of 0.1 mg/mL in 1× final concentration SDS-PAGEloading buffer (4× Laemmli Sample Buffer Bio-Rad #1610747) with 0.1 mMDTT final added freshly. Samples were incubated as 50-500 uL aliquots intightly sealed container (1.7 mL Eppendorf tube) at 95° C. for 10minutes to denature protein. Heated samples were clarified at 20,000×g,25° C. for 5 minutes prior to resolving on a gradient (e.g., 4-10%polyacrylamide) SDS-PAGE gel (Bio-Rad Criterion gel, cat #5671091)according to the manufacturer's instructions. Between 100 ng-5 ugprotein was loaded per gel lane depending on staining method. Inadjacent lanes, molecular weight markers covering range 10-200 kDa(Bio-Rad Precision Plus markers, cat #1610373) were loaded according tothe manufacturer's recommendations. In one example, protein bands werevisualized using Bio-Rad QC Colloidal Coomassie Stain (cat #1610803)according to the manufacturer's instructions.

Destained gels were scanned before measuring band intensity using theBio-Rad Gel Doc system equipped with Image Lab (cat #1708270EDU). Bandswere detected, quantified and sized using automated band detection andmolecular weight calibration against the standard loaded. Data wasexported to Microsoft Excel. Individual band intensity was summed todemonstrate that more than 50% of individual band intensities residedbetween 10 kDa and 200 kDa.

Example 7 Measurement of Change in Buffering Capacity Before and afterProcess Variant

Two independent pilot-scale process replicates of protein compositionsderived from the beginning (“Lysate”) and end (“Final Product”) ProcessVariant C (FIG. 2 ) using S. cerevisiae cells as starting material wereobtained as freeze-dried powders. Suspensions (500 mL) were made of eachreplicate to 2% (w/v) final suspension, transferring 200 mL to a glassbeaker equipped with a magnetic stir bar. The suspension was mixed well(200 rpm-700 rpm). Suspension pH was measured using a pH meter equippedwith pH probe (ThermoFisher Orion module cat #VSTAR82) calibratedagainst standardized buffer solutions (ThermoFisher cat #810199).

In 0.2 mL increments, 5M NaOH was added until pH=12.0+/−0.1 unit. Totalvolume of NaOH solution added was recorded, then the sample wasdiscarded. The process was repeated with a fresh aliquot of 200 mLsuspension, except increments of 5M HCl were added in 0.2 mL incrementsuntil pH=3.0+/−0.1 unit. Total volume of HCl solution added wasrecorded, then the sample was discarded.

To compute buffering capacity of each suspension, total volumes of HCland NaOH added during titration were summed and expressed in milliliters(mL). This number was multiplied by 5 to obtain total millimoles (mmol)NaOH required to adjust 200 mL of the 2% (w/v) solution (4 grams drysolids) from a starting pH of 3.0 to a final pH of 12.0. Using thismethod, a protein composition (Final Product) required only 2.5 mmol pergram dry solids, whereas the starting material (Lysate) required about50% more NaOH to achieve the same shift in pH.

Example 8 Measure of Hydrogen Sulfide Release Capacity of ProteinComposition

Freeze-dried final product of Process Variants B or C (FIG. 2 ), from S.cerevisiae, P. pastoris or E. coli, were suspended to 2% (w/v) inMILLIQ® water and adjusted to a final pH of about 7.0 using HCl, usingthe method as described above for measuring buffer capacity. A volume of5 mL of the pH-adjusted sample was transferred to a 50 mL Falcon conicaltube (Corning cat #352070). Triplicate samples were made, either with 25mM final concentration of L-cysteine (Acros cat #173600250) or with anequivalent volume of water added (control). Into the cap of each tubewas fitted a single filter from the Hach kit for detecting hydrogensulfide (Hach HS—C cat #2537900). Capped tubes were left at roomtemperature (25° C.) for about 24 hours to detect released hydrogensulfide.

A standard curve was used to determine hydrogen sulfide released, asfollows. The Hach detection kit used provides a reference set of imagesfor use in determining hydrogen sulfide (expressed as ppm). Thecolorimetric intensity of these images was measured using a KonicaMinolta Chroma Meter CR-5 E350, with each reference image measuredagainst a black background. This produced a standard curve linear in the“b” channel (yellow) between 0 and 1 ppm hydrogen sulfide. To determinehydrogen sulfide release in the four unknown samples (E. coli (VariantC), Pichia (Variant B), Saccharomyces (Variant C), and Saccharomyces(Variant B)), filter colors were measured with same device, and the “b”(yellow) intensity compared to the standard curve. Results of thisassay, given in FIG. 6 , show cysteine-dependent increase in hydrogensulfide release. Control experiments (FIGS. 6 and 7 ) demonstrated thatthis activity: 1) decreased from high (>1 ppm) in starting lysates of S.cerevisiae with no added L-cysteine to undetectable in final products ofProcess Variants B or C; 2) was inhibited by pyridoxal hydrochloride,consistent with this being a characterized sulfhydrylase enzyme(Yamagata and Takeshima (1976) J. Biochem 80: 777). In the experimentshown in FIG. 7 , lysates from Saccharomyces cells were prepared atabout 2% (w/v) final by taking an in-process sample after homogenizerlysis during Process Variant C, quantifying dry solids, thensupplementing with water (control), or to 50 mM final pyridoxalhydrochloride (pH 7) or 50 mM final cysteine (pH 7). Sulfide release wasmeasured as described, using HS—C sulfide detection filters andcomparing to the kit standard given (shown in FIG. 7 ). Note thatpyridoxal inhibits sulfide release, while non-diafiltered lysate yieldsa strong sulfide signal that is further increased upon addition ofL-cysteine.

Example 9 Measure Rheology of Lysates, Centrates and Final ProductProtein Compositions During Heating

Freeze-dried lysates, centrates or final products of Process Variants Bor C, from S. cerevisiae or P. pastoris, were suspended to 10% (w/v) inMILLI-Q® water and adjusted to a final pH of about 7.0 using NaOH orHCl, using the method as described above for measuring buffer capacity.A volume of 1.25 mL of pH-adjusted sample was transferred to a steel(Peltier) plate in a hybrid rheometer (TA instruments, DHR unit).Storage modulus was measured following manufacturer's recommendationswhile temperature was increased at a ramp rate of 3° C. per minute from25° C. to 95° C. Resulting storage modulus data was plotted in log scaleagainst corresponding temperature in linear scale to yield FIGS. 3, 4and 5 .

Example 10 Measure Thermal Exposure of Hydrophobic Amino Acids

Freeze-dried lysates, centrates or final products of Process Variants Bor C were prepared as suspensions at various pH (e.g., pH 6, 7, 8, 9)and concentrations (e.g. 0.5%, 1%, 2% w/v). The relative and totalfluorescent signal increase during thermal denaturation was measuredusing the “Thermal Shift” method as described in Lo et al (2004) Anal.Biochem. 332(1):153. Samples were run on a Bio-Rad CFX96 (C1000 Touch)device using factory calibrations. Data from Channel 2 (HEX) wereplotted as relative fluorescence intensity (RFI) vs. temperature.Fluorescence was read once per minute during a 1° C. per minute ramprate from 25° C. to 100° C. After subtracting baseline and dye-onlysignal, maximal fluorescence was assigned as maximum peak height tocalculate hydrophobic exposure). Maximal peak height was taken as ‘100%denaturation’ of a sample.

Example 11 Measurement of Particle Size Distribution (PSD)

Suspensions of S. cerevisiae or P. pastoris lysates or final product(Process Variant B or C) (FIG. 2 ) were prepared at 10% (w/v). Thesewere dispersed in a Malvern Mastersizer 3000 unit equipped with a HydroMV unit until obscuration reached about 15%. Distributions were measuredusing the following instrument parameters. Material properties:refractive index 1.45; Absorbance index 0.001; density 1 g/cm³;Disperant: water; refractive index 1.33; non-spherical particles;background measurement time 10 seconds; sample measurement time 10seconds; obscuration limits 0.1%-50%; ultrasound power, 50%; stirrer,2000 rpm. Using these values and Process Variants B and C, PSD valueswere observed as given in the Table 2: D10<0.1 μm; D50<1.0 μm; D90<5 μm.Characteristic PSD values were considerably larger when pH 6 process wasused, presumably due to electrostatic-mediated aggregation.

Example 12 Sampling and Characterization of Headspace Profile forMicrobial Protein Samples

In-process samples prepared from microbial extracts of S. cerevisiae, P.pastoris and E. coli (LY, CN, MF retentate, MF permeate, UF retentate,UF permeate, final product) prepared at process pH of 6.0-6.5 (control)or pH 9.0-9.5 (test) were all made to 10% solutions (wt/wt) withMILLI-Q® water. All samples were adjusted to pH 6.5 (or pH 9.5) with 10MNaOH or 3 M HCl. Three mL of each sample were measured into 20 mL GCMSvials. To evaluate the production of volatile compounds, an Agilent7890A GC coupled with a Leco Pegasus HT-C High Throughput TOFMS was usedalong with a Gerstel MultiPurpose Sampler for auto-sampling. The Gerstelauto-sampler was used perform HS-SPME on each of the samples. Eachsample was incubated at 50° C. for 15 mins with 250 rpm agitation beforebeing extracted for 20 minutes at 50° C. with 250 rpm agitation using a2 cm 50/30 μm Divinylbenzene/Carboxen/Polydimethylsiloxane(DVB/CAR/PDMS), Stable flex, 23 Ga, Autosampler (Supleco, Cat #57299-U)SPME fiber.

Extracted samples were run on the GCMS by desorbing the SPME fiber intoa Gerstel septum-less head with a PTV inlet set to 240° C. for 60seconds and cryofocused at −50° C. with a Gerstel Cooled InjectionSystem (CIS). The CIS was held at −50° C. for 0.1 min, temperature wasramped at +12° C./second to 240° C., and held for the remainder of therun. The desorbed sample was separated on a 60-meter wax column(Agilent, VF-WAXms 60 m×0.25 mm×0.25 mm, Part #CP9207) using a 50 minuteGC method (35° C. for 2 minutes, ramp of 5° C./min until 255° C., holdat 255° C. for 4 minutes) and 1.5 mL/min helium flow rate in splitlessmode. Separated compounds were analyzed by the mass spectrometer, andall data was collected over a 20-500μ mass range, with an acquisitionrate of 10 spectra/sec, and a detector voltage off-set of 200.

Samples were then analyzed using Leco ChromaTOF optimized for Pegasus 4D(Version 4.71.0.0) coupled with the NIST MS Search 2.2. The identity ofeach peak in each sample was identified in a two-step process. First,the mass spectrum of each peak with a signal to noise greater than 30was matched to a mass spectrum in the NIST library using a similaritythreshold of 650. Additionally, an internally developed calibrationmethod was applied to the data set to confirm the identity of thecompounds of interest. In the second step, the Statistical Comparefunction of ChromaTOF was used to align the named analyses across allsamples in a set. The criteria for aligning a single peak across allsamples were a match score (similarity between the peak spectra acrossall samples) of 700, a maximum retention time difference of 10 seconds,and the peak must have been present in at least two samples.

Table 4 includes compounds assayed using this technique.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A protein composition produced by a methodcomprising: a) washing an aqueous suspension of a plurality of cells toyield a washed aqueous suspension; b) lysing the washed aqueoussuspension of the plurality of cells to obtain a cell lysate; and c)filtering the cell lysate to obtain the protein composition; whereinsteps a), b), and c) independently, are performed at a pH between about8.5 and about 12.0.
 2. The protein composition of claim 1, whereinfiltering comprises microfiltration ultrafiltration, diafiltration, or acombination thereof.
 3. The protein composition of claim 1, furthercomprising clarifying the cell lysate, to obtain a clarified lysate. 4.The protein composition of claim 3, wherein the clarifying is performedby centrifugation to less than about 20% dry solids.
 5. The proteincomposition of claim 1, further comprising pasteurizing the proteincomposition to obtain a pasteurized protein composition, wherein thepasteurizing is performed at a pH between about 8.5 and about 12.0. 6.The protein composition of claim 1, wherein the lysing is performedbiochemically or chemically.
 7. A protein composition produced by amethod comprising: a) lysing an aqueous suspension of a plurality ofcells to obtain a cell lysate; and b) filtering the cell lysate toobtain the protein composition; wherein steps a) and b) independently,are performed at a pH between about 8.5 and about 12.0, and wherein theprotein composition has a buffering capacity of less than about 2.5 mmolNaOH per gram dry solids.
 8. The protein composition of claim 7, whereinthe protein composition has a buffering capacity of less than about 2.0mmol NaOH per gram dry solids.
 9. The protein composition of claim 7,wherein the lysing is performed biochemically or chemically.
 10. Aprotein composition produced by a method comprising: a) lysing anaqueous suspension of a plurality of cells to obtain a cell lysate; andb) filtering the cell lysate to obtain the protein composition; whereinsteps a) and b) independently, are performed at a pH between about 8.5and about 12.0, and wherein H₂S is detectable in an amount of less thanabout 0.1 ppm after about 24 hours at 25° C. when L-cysteine is notadded to 5 mL of a 2% (w/v) suspension of the protein composition at pH7.0 and/or wherein H₂S is detectable in an amount of at least about 0.2ppm about 24 hours at 25° C. after 5 mL of a 2% (w/v) suspension of theprotein composition is brought to about 25 mM final concentration ofL-cysteine.
 11. The protein composition of claim 10, wherein at leastabout 50% of the protein in the protein composition falls between about10 kDa and about 200 kDa.
 12. The protein composition of claim 10,wherein the plurality of cells comprises microbial cells.
 13. Theprotein composition of claim 10, wherein the protein compositioncomprises at least about 35%, on a dry weight basis, of compounds largerthan 5 kDa.
 14. The protein composition of claim 10, wherein theplurality of cells comprises fungal cells.
 15. The protein compositionof claim 14, wherein the fungal cells are selected from the groupconsisting of Saccharomyces, Pichia, Candida, Hansenula, Torulopsis,Kluyveromyces, Yarrowia, and Fusarium cells.
 16. The protein compositionof claim 10, wherein the plurality of cells comprises bacterial cells.17. The protein composition of claim 16, wherein the bacterial cells areselected from the group consisting of Bacillus, Escherichia,Lactobacillus, Corynebacterium, Pseudomonas, and Methanococcus.
 18. Theprotein composition of claim 10, wherein H₂S is detectable in an amountof less than about 0.1 ppm about 24 hours at 25° C. when L-cysteine isnot added to 5 mL of a 2% (w/v) suspension of the protein composition atpH 7.0, and wherein H₂S is detectable in an amount of at least about 0.2ppm about 24 hours at 25° C. after 5 mL of a 2% (w/v) suspension of theprotein composition is brought to about 25 mM final concentration ofL-cysteine.
 19. The protein composition of claim 10, wherein H₂S isdetectable in an amount of at least about 0.3 ppm about 24 hours at 25°C. when 5 mL of a 2% (w/v) suspension of the protein composition isbrought to about 25 mM final concentration of L-cysteine.
 20. Theprotein composition of claim 10, wherein the lysing is performedbiochemically or chemically.